技术领域Technical Field
本发明涉及眼科材料制备技术领域，具体涉及一种脱细胞羊膜材料及其制备方法与在制备眼表修复材料中的应用。The present invention relates to the technical field of ophthalmic material preparation, and in particular to a decellularized amniotic membrane material and a preparation method thereof, and application thereof in the preparation of an ocular surface repair material.
背景技术Background technique
羊膜是单层上皮细胞互相连接构成的薄膜；是胎膜最内一层，是一层半透明的薄膜，与覆盖在胎盘、脐带的羊膜层相连接。在眼科临床中，羊膜可以作为一种生物材料用于治疗眼表损伤；具有重要的应用价值。The amniotic membrane is a thin membrane composed of a single layer of epithelial cells connected to each other. It is the innermost layer of the fetal membrane and is a translucent membrane that is connected to the amniotic membrane covering the placenta and umbilical cord. In ophthalmology clinics, the amniotic membrane can be used as a biomaterial to treat ocular surface injuries and has important application value.
如中国发明专利CN202210366231.9公开了一种脱细胞冻干羊膜产品的制备工艺，通过对胎盘剥离、分型，对羊膜进行化学处理、清洗、脱细胞、再清洗、冷冻干燥、切割成型、内包装、辐照灭菌、外包成品的工艺，针对有绒毛层和无绒毛层两种羊膜型号，能够有效进行脱细胞，且充分考虑到羊膜透明、体积小等不易于换液以及冷冻干燥后尺寸难精准控制的关键点，合理规划生产工艺，提高羊膜的成品率，促进羊膜产业发展。For example, Chinese invention patent CN202210366231.9 discloses a preparation process for a decellularized freeze-dried amniotic membrane product. The process involves placental stripping and typing, chemical treatment of the amniotic membrane, cleaning, decellularization, re-cleaning, freeze-drying, cutting and shaping, inner packaging, irradiation sterilization, and outsourcing of the finished product. The process can effectively decellularize both the amniotic membrane types, with and without a chorionic layer. The process also fully takes into account the key points that the amniotic membrane is transparent, small in size, and difficult to change the fluid, as well as the difficulty in accurately controlling the size after freeze-drying. The production process is rationally planned to improve the yield of the amniotic membrane and promote the development of the amniotic membrane industry.
然而，发明人在研究中发现，参照上述方法制备得到的脱细胞羊膜材料，其促进细胞增殖作用不强，将其用于眼表损伤修复时，修复的周期较长，有待进一步提高。However, the inventors found in their research that the decellularized amniotic membrane material prepared by the above method has a weak effect in promoting cell proliferation. When it is used to repair ocular surface damage, the repair cycle is relatively long and needs to be further improved.
发明内容Summary of the invention
为了解决现有技术中存在的至少之一的技术问题，本发明提供了一种脱细胞羊膜材料及其制备方法与在制备眼表修复材料中的应用。In order to solve at least one of the technical problems existing in the prior art, the present invention provides a decellularized amniotic membrane material, a preparation method thereof, and an application thereof in preparing an ocular surface repair material.
本发明解决上述技术问题的技术方案如下：The technical solution of the present invention to solve the above technical problems is as follows:
本发明首先提供了一种脱细胞羊膜材料的制备方法，其包含如下步骤：The present invention first provides a method for preparing a decellularized amniotic membrane material, which comprises the following steps:
(1)取胎盘，将羊膜从胎盘上进行分离，接着用细胞刮匙去除羊膜表面多余的组织，得无绒毛层羊膜；(1) taking the placenta, separating the amniotic membrane from the placenta, and then using a cell scraper to remove excess tissue on the surface of the amniotic membrane to obtain the amniotic membrane without chorionic villi;
(2)将经过步骤(1)处理后的无绒毛层羊膜进行清洗，得清洗后的无绒毛层羊膜；(2) washing the chorionic villus-free amniotic membrane after being treated in step (1) to obtain a washed chorionic villus-free amniotic membrane;
(3)将清洗后的无绒毛层羊膜放入脱细胞试剂中进行脱细胞处理，得脱细胞羊膜；(3) placing the washed amniotic membrane without chorionic villus layer into a decellularizing reagent for decellularization to obtain a decellularized amniotic membrane;
(4)将脱细胞羊膜进行清洗，接着放入含天然提取物的乙醇溶液中进行浸泡处理；(4) washing the decellularized amniotic membrane and then soaking it in an ethanol solution containing a natural extract;
(5)取出经步骤(4)处理过的脱细胞羊膜，再经干燥、剪切以及辐照灭菌后，得所述的脱细胞羊膜材料。(5) Taking out the decellularized amniotic membrane treated in step (4), and then drying, shearing and irradiating and sterilizing it to obtain the decellularized amniotic membrane material.
本发明提供了一种全新的脱细胞羊膜材料的制备方法；研究表明，在本发明所述的制备方法中，通过将脱细胞羊膜进行天然提取物浸泡处理制备得到的脱细胞羊膜材料，其具有较好的促进细胞增殖作用。The present invention provides a novel method for preparing a decellularized amniotic membrane material. Studies have shown that, in the preparation method of the present invention, the decellularized amniotic membrane material prepared by soaking the decellularized amniotic membrane in natural extracts has a good effect of promoting cell proliferation.
优选地，步骤(1)所述的胎盘在分离羊膜处理前，进行病原体筛查。Preferably, the placenta described in step (1) is screened for pathogens before separation of the amniotic membrane.
优选地，步骤(2)中所述的清洗，是指用含青霉素的生理盐水进行清洗。Preferably, the cleaning in step (2) refers to cleaning with physiological saline containing penicillin.
优选地，所述的含青霉素的生理盐水中青霉素的浓度为0.1～0.3mg/mL。Preferably, the concentration of penicillin in the penicillin-containing saline is 0.1-0.3 mg/mL.
最优选地，所述的含青霉素的生理盐水中青霉素的浓度为0.2mg/mL。Most preferably, the concentration of penicillin in the penicillin-containing saline is 0.2 mg/mL.
优选地，步骤(3)中所述的脱细胞试剂是指含有Triton X-100以及脱氧胆酸钠的水溶液。Preferably, the decellularization reagent in step (3) refers to an aqueous solution containing Triton X-100 and sodium deoxycholate.
优选地，脱细胞试剂中Triton X-100的含量为0.1～1g/mL；脱氧胆酸钠的含量为0.1～1g/mL；Preferably, the content of Triton X-100 in the decellularization reagent is 0.1 to 1 g/mL; the content of sodium deoxycholate is 0.1 to 1 g/mL;
最优选地，脱细胞试剂中Triton X-100的含量为0.5g/mL；脱氧胆酸钠的含量为0.5g/mL。Most preferably, the content of Triton X-100 in the decellularization reagent is 0.5 g/mL; the content of sodium deoxycholate is 0.5 g/mL.
优选地，步骤(4)含天然提取物的水溶液中的天然提取物通过如下方法制备得到：Preferably, the natural extract in the aqueous solution containing the natural extract in step (4) is prepared by the following method:
藏红花和灵芝粉碎后用有机溶剂进行提取，提取结束后取提取液，浓缩干燥后得藏红花-灵芝有机溶剂提取物；Saffron and ganoderma lucidum are crushed and then extracted with an organic solvent. After the extraction, the extract is taken and concentrated and dried to obtain a saffron-ganoderma lucidum organic solvent extract;
取藏红花-灵芝有机溶剂提取物即得所述的天然提取物。The natural extract is obtained by taking the saffron-Ganoderma lucidum organic solvent extract.
优选地，所述的有机溶剂为丙酮。Preferably, the organic solvent is acetone.
优选地，藏红花和灵芝的重量比为4～6:1～3。Preferably, the weight ratio of saffron to ganoderma lucidum is 4-6:1-3.
最优选地，藏红花和灵芝的重量比为5:2。Most preferably, the weight ratio of saffron to ganoderma is 5:2.
优选地，中药藏红花和灵芝的总重量与有机溶剂的用量比为1kg:8～12L。Preferably, the total weight ratio of the Chinese medicinal materials saffron and ganoderma lucidum to the organic solvent is 1 kg: 8 to 12 L.
最优选地，中药藏红花和灵芝的总重量与有机溶剂的用量比为1kg:10L。Most preferably, the ratio of the total weight of the Chinese medicinal materials saffron and ganoderma lucidum to the amount of the organic solvent is 1 kg:10 L.
优选地，所述的天然提取物的制备方法具体还包括如下步骤：Preferably, the method for preparing the natural extract further comprises the following steps:
取藏红花-灵芝有机溶剂提取物，上大孔树脂柱；先用体积分数为26～29％的乙醇水溶液洗脱去除杂质；接着再用体积分数为41～43％的乙醇水溶液洗脱，收集体积分数为41～43％的乙醇水溶液洗脱下来的洗脱液，浓缩干燥后即得所述的天然提取物。Take the saffron-Ganoderma lucidum organic solvent extract and apply it to a macroporous resin column; first use an ethanol aqueous solution with a volume fraction of 26-29% to elute and remove impurities; then use an ethanol aqueous solution with a volume fraction of 41-43% to elute, collect the eluate eluted with the ethanol aqueous solution with a volume fraction of 41-43%, and concentrate and dry to obtain the natural extract.
最优选地，所述的促进组织再生物质的制备方法具体还包括如下步骤：Most preferably, the method for preparing the substance for promoting tissue regeneration further comprises the following steps:
取藏红花-灵芝有机溶剂提取物，上大孔树脂柱；先用体积分数为28％的乙醇水溶液洗脱去除杂质；接着再用体积分数为42％的乙醇水溶液洗脱，收集体积分数为42％的乙醇水溶液洗脱下来的洗脱液，浓缩干燥后即得所述的天然提取物。Take the saffron-Ganoderma lucidum organic solvent extract and apply it to a macroporous resin column; first elute it with a 28% by volume ethanol aqueous solution to remove impurities; then elute it with a 42% by volume ethanol aqueous solution, collect the eluate eluted with the 42% by volume ethanol aqueous solution, and concentrate and dry it to obtain the natural extract.
在本发明所述制备方法中，将脱细胞羊膜采用将藏红花-灵芝有机溶剂提取物物进一步通过本发明上述大孔树脂洗脱条件制备得到的天然提取物进行浸泡处理，相比于采用藏红花-灵芝有机溶剂提取物作为天然提取物进行浸泡处理，可以大幅提高制备得到的脱细胞羊膜材料的促进细胞增殖作用。In the preparation method described in the present invention, the decellularized amniotic membrane is soaked in a natural extract obtained by further subjecting the saffron-Ganoderma lucidum organic solvent extract to the above-mentioned macroporous resin elution conditions of the present invention. Compared with soaking in the saffron-Ganoderma lucidum organic solvent extract as the natural extract, the cell proliferation-promoting effect of the prepared decellularized amniotic membrane material can be greatly improved.
此外，发明人在研究中还发现，本发明的大孔树脂洗脱条件也十分关键；将脱细胞羊膜必须采用将藏红花-灵芝有机溶剂提取物进一步通过本发明上述大孔树脂洗脱条件制备得到的天然提取物进行浸泡处理，相比于采用藏红花-灵芝有机溶剂提取物作为天然提取物进行浸泡处理，才能进一步大幅提高制备得到的脱细胞羊膜材料的促进细胞增殖作用；然而，采用将藏红花-灵芝有机溶剂提取物物进一步通过其它大孔树脂洗脱条件制备得到的天然提取物进行浸泡处理，并不能进一步大幅提高制备得到的脱细胞羊膜材料的促进细胞增殖作用。In addition, the inventors also found in the research that the macroporous resin elution conditions of the present invention are also very critical; the decellularized amniotic membrane must be soaked in a natural extract prepared by further subjecting the saffron-Ganoderma organic solvent extract to the above-mentioned macroporous resin elution conditions of the present invention, compared with soaking in the saffron-Ganoderma organic solvent extract as a natural extract, in order to further significantly improve the cell proliferation promoting effect of the prepared decellularized amniotic membrane material; however, soaking in a natural extract prepared by further subjecting the saffron-Ganoderma organic solvent extract to other macroporous resin elution conditions does not further significantly improve the cell proliferation promoting effect of the prepared decellularized amniotic membrane material.
优选地，骤(4)含天然提取物的乙醇溶液中天然提取物的质量分数为0.5～2％。Preferably, the mass fraction of the natural extract in the ethanol solution containing the natural extract in step (4) is 0.5-2%.
最优选地，骤(4)含天然提取物的乙醇溶液中天然提取物的质量分数为1％。Most preferably, the mass fraction of the natural extract in the ethanol solution containing the natural extract in step (4) is 1%.
优选地，骤(4)含天然提取物的乙醇溶液中的乙醇溶液是指体积分数为75％的乙醇溶液。Preferably, the ethanol solution in the ethanol solution containing the natural extract in step (4) refers to an ethanol solution with a volume fraction of 75%.
优选地，步骤(4)中的浸泡处理的时间为3～6h。Preferably, the soaking treatment time in step (4) is 3 to 6 hours.
最优选地，步骤(4)中的浸泡处理的时间为5h。Most preferably, the soaking treatment time in step (4) is 5 hours.
本发明还提供了一种上述制备方法制备得到的脱细胞羊膜材料。The present invention also provides a decellularized amniotic membrane material prepared by the above preparation method.
本发明还提供了一种上述脱细胞羊膜材料在制备眼表修复材料中的应用。The present invention also provides a use of the above-mentioned decellularized amniotic membrane material in preparing an ocular surface repair material.
有益效果：本发明提供了一种全新的脱细胞羊膜材料的制备方法；研究表明，在本发明所述的制备方法中，通过将脱细胞羊膜进行天然提取物浸泡处理制备得到的脱细胞羊膜材料，其具有较好的促进细胞增殖作用。因此，将本发明制备得到的脱细胞羊膜材料用于眼表损伤修复时，有助于缩短修复周期，具有重要的应用价值。Beneficial effects: The present invention provides a new method for preparing decellularized amniotic membrane materials; studies have shown that in the preparation method of the present invention, the decellularized amniotic membrane material prepared by soaking the decellularized amniotic membrane in natural extracts has a good effect of promoting cell proliferation. Therefore, when the decellularized amniotic membrane material prepared by the present invention is used for repairing ocular surface damage, it helps to shorten the repair cycle and has important application value.
具体实施方式Detailed ways
以下结合具体实施例来进一步解释本发明，但实施例并不限定本发明的保护范围。The present invention is further explained below in conjunction with specific examples, but the examples do not limit the scope of protection of the present invention.
实施例1脱细胞羊膜材料的制备方法Example 1 Preparation method of decellularized amniotic membrane material
(1)取经病原体筛查后的离体的人体胎盘，将羊膜从胎盘上进行分离，接着用细胞刮匙去除羊膜表面多余的组织，得无绒毛层羊膜；(1) obtaining an isolated human placenta after pathogen screening, separating the amnion from the placenta, and then removing excess tissue on the surface of the amnion with a cell scraper to obtain a chorionic amnion;
(2)将经过步骤(1)处理后的无绒毛层羊膜进行清洗，得清洗后的无绒毛层羊膜；所述的清洗是指采用青霉素的浓度为0.2mg/mL的含青霉素的生理盐水进行清洗；(2) washing the chorionic amniotic membrane treated in step (1) to obtain a washed chorionic amniotic membrane; the washing refers to washing with a penicillin-containing saline solution having a penicillin concentration of 0.2 mg/mL;
(3)将清洗后的无绒毛层羊膜放入脱细胞试剂中进行脱细胞振荡处理40min，得脱细胞羊膜；所述的脱细胞试剂是指含有Triton X-100以及脱氧胆酸钠的水溶液，其中，Triton X-100的含量为0.5g/mL；脱氧胆酸钠的含量为0.5g/mL；(3) placing the cleaned chorionic villus-free amniotic membrane in a decellularization reagent for decellularization oscillation treatment for 40 minutes to obtain a decellularized amniotic membrane; the decellularization reagent refers to an aqueous solution containing Triton X-100 and sodium deoxycholate, wherein the content of Triton X-100 is 0.5 g/mL; the content of sodium deoxycholate is 0.5 g/mL;
(4)将脱细胞羊膜进行清洗，接着放入含天然提取物的乙醇溶液中进行浸泡处理5h；其中，含天然提取物的乙醇溶液中天然提取物的质量分数为1％，所述的乙醇溶液是指体积分数为75％的乙醇溶液；(4) washing the decellularized amniotic membrane, and then soaking it in an ethanol solution containing a natural extract for 5 hours; wherein the mass fraction of the natural extract in the ethanol solution containing the natural extract is 1%, and the ethanol solution refers to an ethanol solution with a volume fraction of 75%;
(5)取出经步骤(4)处理过的脱细胞羊膜，再经干燥、剪切以及辐照灭菌后，得所述的脱细胞羊膜材料；(5) taking out the decellularized amniotic membrane treated in step (4), and then drying, shearing and irradiating and sterilizing it to obtain the decellularized amniotic membrane material;
其中，步骤(4)中所述的天然提取物通过如下方法制备得到：Wherein, the natural extract described in step (4) is prepared by the following method:
取中藏红花和灵芝粉碎后用有机溶剂在56℃下进行回流提取1h，提取结束后取提取液，浓缩干燥后得藏红花-灵芝有机溶剂提取物；其中，藏红花和灵芝的重量比为5:2；中药藏红花和灵芝的总重量与有机溶剂的用量比为1kg:10L；所述的有机溶剂为丙酮；Saffron and ganoderma lucidum are crushed and refluxed with an organic solvent at 56° C. for 1 hour. After the extraction, the extract is taken and concentrated and dried to obtain a saffron-ganoderma lucidum organic solvent extract; wherein the weight ratio of saffron and ganoderma lucidum is 5:2; the total weight ratio of saffron and ganoderma lucidum to the amount of the organic solvent is 1 kg:10 L; the organic solvent is acetone;
取藏红花-灵芝有机溶剂提取物即得所述的天然提取物。The natural extract is obtained by taking the saffron-Ganoderma lucidum organic solvent extract.
实施例2脱细胞羊膜材料的制备方法Example 2 Preparation method of decellularized amniotic membrane material
(1)取经病原体筛查后的离体的人体胎盘，将羊膜从胎盘上进行分离，接着用细胞刮匙去除羊膜表面多余的组织，得无绒毛层羊膜；(1) obtaining an isolated human placenta after pathogen screening, separating the amnion from the placenta, and then removing excess tissue on the surface of the amnion with a cell scraper to obtain a chorionic amnion;
(2)将经过步骤(1)处理后的无绒毛层羊膜进行清洗，得清洗后的无绒毛层羊膜；所述的清洗是指采用青霉素的浓度为0.2mg/mL的含青霉素的生理盐水进行清洗；(2) washing the chorionic amniotic membrane treated in step (1) to obtain a washed chorionic amniotic membrane; the washing refers to washing with a penicillin-containing saline solution having a penicillin concentration of 0.2 mg/mL;
(3)将清洗后的无绒毛层羊膜放入脱细胞试剂中进行脱细胞振荡处理40min，得脱细胞羊膜；所述的脱细胞试剂是指含有Triton X-100以及脱氧胆酸钠的水溶液，其中，Triton X-100的含量为0.5g/mL；脱氧胆酸钠的含量为0.5g/mL；(3) placing the cleaned chorionic villus-free amniotic membrane in a decellularization reagent for decellularization oscillation treatment for 40 minutes to obtain a decellularized amniotic membrane; the decellularization reagent refers to an aqueous solution containing Triton X-100 and sodium deoxycholate, wherein the content of Triton X-100 is 0.5 g/mL; the content of sodium deoxycholate is 0.5 g/mL;
(4)将脱细胞羊膜进行清洗，接着放入含天然提取物的乙醇溶液中进行浸泡处理5h；其中，含天然提取物的乙醇溶液中天然提取物的质量分数为1％，所述的乙醇溶液是指体积分数为75％的乙醇溶液；(4) washing the decellularized amniotic membrane, and then soaking it in an ethanol solution containing a natural extract for 5 hours; wherein the mass fraction of the natural extract in the ethanol solution containing the natural extract is 1%, and the ethanol solution refers to an ethanol solution with a volume fraction of 75%;
(5)取出经步骤(4)处理过的脱细胞羊膜，再经干燥、剪切以及辐照灭菌后，得所述的脱细胞羊膜材料；(5) taking out the decellularized amniotic membrane treated in step (4), and then drying, shearing and irradiating and sterilizing it to obtain the decellularized amniotic membrane material;
其中，步骤(4)中所述的天然提取物通过如下方法制备得到：Wherein, the natural extract described in step (4) is prepared by the following method:
(1)取中藏红花和灵芝粉碎后用有机溶剂在56℃下进行回流提取1h，提取结束后取提取液，浓缩干燥后得藏红花-灵芝有机溶剂提取物；其中，藏红花和灵芝的重量比为5:2；中药藏红花和灵芝的总重量与有机溶剂的用量比为1kg:10L；所述的有机溶剂为丙酮；(1) Saffron and Ganoderma lucidum are crushed and then refluxed with an organic solvent at 56° C. for 1 hour. After the extraction, the extract is taken and concentrated and dried to obtain a saffron-Ganoderma lucidum organic solvent extract; wherein the weight ratio of saffron to Ganoderma lucidum is 5:2; the total weight ratio of saffron and Ganoderma lucidum to the organic solvent is 1 kg:10 L; and the organic solvent is acetone;
(2)取藏红花-灵芝有机溶剂提取物，上HP-20型大孔树脂柱；先用5倍柱体积的体积分数为28％的乙醇水溶液洗脱去除杂质；接着再用5倍柱体积的体积分数为42％的乙醇水溶液洗脱，收集体积分数为42％的乙醇水溶液洗脱下来的洗脱液，浓缩干燥后即得所述的天然提取物。(2) Take the saffron-Ganoderma lucidum organic solvent extract and apply it to an HP-20 macroporous resin column; first use 5 times the column volume of 28% ethanol aqueous solution to elute and remove impurities; then use 5 times the column volume of 42% ethanol aqueous solution to elute, collect the eluate eluted with 42% ethanol aqueous solution, and concentrate and dry to obtain the natural extract.
对比例1脱细胞羊膜材料的制备方法Comparative Example 1 Preparation method of decellularized amniotic membrane material
(1)取经病原体筛查后的离体的人体胎盘，将羊膜从胎盘上进行分离，接着用细胞刮匙去除羊膜表面多余的组织，得无绒毛层羊膜；(1) obtaining an isolated human placenta after pathogen screening, separating the amnion from the placenta, and then removing excess tissue on the surface of the amnion with a cell scraper to obtain a chorionic amnion;
(2)将经过步骤(1)处理后的无绒毛层羊膜进行清洗，得清洗后的无绒毛层羊膜；所述的清洗是指采用青霉素的浓度为0.2mg/mL的含青霉素的生理盐水进行清洗；(2) washing the chorionic amniotic membrane treated in step (1) to obtain a washed chorionic amniotic membrane; the washing refers to washing with a penicillin-containing saline solution having a penicillin concentration of 0.2 mg/mL;
(3)将清洗后的无绒毛层羊膜放入脱细胞试剂中进行脱细胞振荡处理40min，得脱细胞羊膜；所述的脱细胞试剂是指含有Triton X-100以及脱氧胆酸钠的水溶液，其中，Triton X-100的含量为0.5g/mL；脱氧胆酸钠的含量为0.5g/mL；(3) placing the cleaned chorionic villus-free amniotic membrane in a decellularization reagent for decellularization oscillation treatment for 40 minutes to obtain a decellularized amniotic membrane; the decellularization reagent refers to an aqueous solution containing Triton X-100 and sodium deoxycholate, wherein the content of Triton X-100 is 0.5 g/mL; the content of sodium deoxycholate is 0.5 g/mL;
(4)将脱细胞羊膜进行清洗，接着放入含天然提取物的乙醇溶液中进行浸泡处理5h；其中，含天然提取物的乙醇溶液中天然提取物的质量分数为1％，所述的乙醇溶液是指体积分数为75％的乙醇溶液；(4) washing the decellularized amniotic membrane, and then soaking it in an ethanol solution containing a natural extract for 5 hours; wherein the mass fraction of the natural extract in the ethanol solution containing the natural extract is 1%, and the ethanol solution refers to an ethanol solution with a volume fraction of 75%;
(5)取出经步骤(4)处理过的脱细胞羊膜，再经干燥、剪切以及辐照灭菌后，得所述的脱细胞羊膜材料；(5) taking out the decellularized amniotic membrane treated in step (4), and then drying, shearing and irradiating and sterilizing it to obtain the decellularized amniotic membrane material;
其中，步骤(4)中所述的天然提取物通过如下方法制备得到：Wherein, the natural extract described in step (4) is prepared by the following method:
(1)取中藏红花和灵芝粉碎后用有机溶剂在56℃下进行回流提取1h，提取结束后取提取液，浓缩干燥后得藏红花-灵芝有机溶剂提取物；其中，藏红花和灵芝的重量比为5:2；中药藏红花和灵芝的总重量与有机溶剂的用量比为1kg:10L；所述的有机溶剂为丙酮；(1) Saffron and Ganoderma lucidum are crushed and then refluxed with an organic solvent at 56° C. for 1 hour. After the extraction, the extract is taken and concentrated and dried to obtain a saffron-Ganoderma lucidum organic solvent extract; wherein the weight ratio of saffron to Ganoderma lucidum is 5:2; the total weight ratio of saffron and Ganoderma lucidum to the organic solvent is 1 kg:10 L; and the organic solvent is acetone;
(2)取藏红花-灵芝有机溶剂提取物，上HP-20型大孔树脂柱；先用5倍柱体积的体积分数为10％的乙醇水溶液洗脱去除杂质；接着再用5倍柱体积的体积分数为30％的乙醇水溶液洗脱，收集体积分数为30％的乙醇水溶液洗脱下来的洗脱液，浓缩干燥后即得所述的天然提取物。(2) Take the saffron-Ganoderma lucidum organic solvent extract and apply it to an HP-20 macroporous resin column; first use 5 times the column volume of a 10% ethanol aqueous solution to elute and remove impurities; then use 5 times the column volume of a 30% ethanol aqueous solution to elute, collect the eluate eluted with the 30% ethanol aqueous solution, and concentrate and dry to obtain the natural extract.
对比例2脱细胞羊膜材料的制备方法Comparative Example 2 Preparation method of decellularized amniotic membrane material
(1)取经病原体筛查后的离体的人体胎盘，将羊膜从胎盘上进行分离，接着用细胞刮匙去除羊膜表面多余的组织，得无绒毛层羊膜；(1) obtaining an isolated human placenta after pathogen screening, separating the amnion from the placenta, and then removing excess tissue on the surface of the amnion with a cell scraper to obtain a chorionic amnion;
(2)将经过步骤(1)处理后的无绒毛层羊膜进行清洗，得清洗后的无绒毛层羊膜；所述的清洗是指采用青霉素的浓度为0.2mg/mL的含青霉素的生理盐水进行清洗；(2) washing the chorionic amniotic membrane treated in step (1) to obtain a washed chorionic amniotic membrane; the washing refers to washing with a penicillin-containing saline solution having a penicillin concentration of 0.2 mg/mL;
(3)将清洗后的无绒毛层羊膜放入脱细胞试剂中进行脱细胞振荡处理40min，得脱细胞羊膜；所述的脱细胞试剂是指含有Triton X-100以及脱氧胆酸钠的水溶液，其中，Triton X-100的含量为0.5g/mL；脱氧胆酸钠的含量为0.5g/mL；(3) placing the cleaned chorionic villus-free amniotic membrane in a decellularization reagent for decellularization oscillation treatment for 40 minutes to obtain a decellularized amniotic membrane; the decellularization reagent refers to an aqueous solution containing Triton X-100 and sodium deoxycholate, wherein the content of Triton X-100 is 0.5 g/mL; the content of sodium deoxycholate is 0.5 g/mL;
(4)将脱细胞羊膜进行清洗，接着放入含天然提取物的乙醇溶液中进行浸泡处理5h；其中，含天然提取物的乙醇溶液中天然提取物的质量分数为1％，所述的乙醇溶液是指体积分数为75％的乙醇溶液；(4) washing the decellularized amniotic membrane, and then soaking it in an ethanol solution containing a natural extract for 5 hours; wherein the mass fraction of the natural extract in the ethanol solution containing the natural extract is 1%, and the ethanol solution refers to an ethanol solution with a volume fraction of 75%;
(5)取出经步骤(4)处理过的脱细胞羊膜，再经干燥、剪切以及辐照灭菌后，得所述的脱细胞羊膜材料；(5) taking out the decellularized amniotic membrane treated in step (4), and then drying, shearing and irradiating and sterilizing it to obtain the decellularized amniotic membrane material;
其中，步骤(4)中所述的天然提取物通过如下方法制备得到：Wherein, the natural extract described in step (4) is prepared by the following method:
(1)取中藏红花和灵芝粉碎后用有机溶剂在56℃下进行回流提取1h，提取结束后取提取液，浓缩干燥后得藏红花-灵芝有机溶剂提取物；其中，藏红花和灵芝的重量比为5:2；中药藏红花和灵芝的总重量与有机溶剂的用量比为1kg:10L；所述的有机溶剂为丙酮；(1) Saffron and Ganoderma lucidum are crushed and then refluxed with an organic solvent at 56° C. for 1 hour. After the extraction, the extract is taken and concentrated and dried to obtain a saffron-Ganoderma lucidum organic solvent extract; wherein the weight ratio of saffron to Ganoderma lucidum is 5:2; the total weight ratio of saffron and Ganoderma lucidum to the organic solvent is 1 kg:10 L; and the organic solvent is acetone;
(2)取藏红花-灵芝有机溶剂提取物，上HP-20型大孔树脂柱；先用5倍柱体积的体积分数为40％的乙醇水溶液洗脱去除杂质；接着再用5倍柱体积的体积分数为60％的乙醇水溶液洗脱，收集体积分数为60％的乙醇水溶液洗脱下来的洗脱液，浓缩干燥后即得所述的天然提取物。(2) Take the saffron-Ganoderma lucidum organic solvent extract and apply it to an HP-20 macroporous resin column; first use 5 times the column volume of 40% ethanol aqueous solution to elute and remove impurities; then use 5 times the column volume of 60% ethanol aqueous solution to elute, collect the eluate eluted with 60% ethanol aqueous solution, and concentrate and dry to obtain the natural extract.
实施例1～2以及对比例1～2制备得到的脱细胞羊膜材料促进表皮细胞的增值率见表1。The proliferation rates of epidermal cells promoted by the decellularized amniotic membrane materials prepared in Examples 1-2 and Comparative Examples 1-2 are shown in Table 1.
表1.脱细胞羊膜材料促进表皮细胞的增值率Table 1. Proliferation rate of epidermal cells promoted by acellular amniotic membrane materials
从表1实验结果中可以看出，实施例1制备得到的脱细胞羊膜材料，其促进表皮细胞的增值率为260％；这说明：在本发明所述的制备方法中，通过将脱细胞羊膜经本发明天然提取物浸泡处理后制备得到的脱细胞羊膜材料，其具有较好的促进细胞增殖作用。It can be seen from the experimental results in Table 1 that the decellularized amniotic membrane material prepared in Example 1 has a proliferation rate of epidermal cells of 260%. This indicates that in the preparation method described in the present invention, the decellularized amniotic membrane material prepared by soaking the decellularized amniotic membrane with the natural extract of the present invention has a better effect in promoting cell proliferation.
从表1实验结果中可以看出，实施例2制备得到的脱细胞羊膜材料，其促进表皮细胞的增值率为540％，大幅高于实施例1制备得到的脱细胞羊膜材料；这说明：在本发明所述制备方法中，将脱细胞羊膜采用将藏红花-灵芝有机溶剂提取物物进一步通过本发明上述大孔树脂洗脱条件制备得到的天然提取物进行浸泡处理，相比于采用藏红花-灵芝有机溶剂提取物作为天然提取物进行浸泡处理，可以大幅提高制备得到的脱细胞羊膜材料的促进细胞增殖作用。It can be seen from the experimental results in Table 1 that the decellularized amniotic membrane material prepared in Example 2 has a proliferation rate of epidermal cells of 540%, which is significantly higher than that of the decellularized amniotic membrane material prepared in Example 1. This shows that in the preparation method described in the present invention, the decellularized amniotic membrane is soaked in a natural extract obtained by further eluting the saffron-Ganoderma lucidum organic solvent extract under the above-mentioned macroporous resin elution conditions of the present invention. Compared with soaking in the saffron-Ganoderma lucidum organic solvent extract as a natural extract, the cell proliferation promoting effect of the prepared decellularized amniotic membrane material can be greatly improved.
从表1实验结果中可以看出，对比例1制备得到的脱细胞羊膜材料，其促进表皮细胞的增值率与实施例1相比虽然有提高，但是提高幅度不大；对比例2制备得到的脱细胞羊膜材料，其促进表皮细胞的增值率与实施例1相比不但没有提高，反而降低了；这说明：本发明的大孔树脂洗脱条件也十分关键；将脱细胞羊膜必须采用将藏红花-灵芝有机溶剂提取物进一步通过本发明上述大孔树脂洗脱条件制备得到的天然提取物进行浸泡处理，相比于采用藏红花-灵芝有机溶剂提取物作为天然提取物进行浸泡处理，才能进一步大幅提高制备得到的脱细胞羊膜材料的促进细胞增殖作用；然而，采用将藏红花-灵芝有机溶剂提取物物进一步通过其它大孔树脂洗脱条件制备得到的天然提取物进行浸泡处理，并不能进一步大幅提高制备得到的脱细胞羊膜材料的促进细胞增殖作用。It can be seen from the experimental results in Table 1 that the decellularized amniotic membrane material prepared in Comparative Example 1 has an improved rate of promoting the proliferation of epidermal cells compared with that in Example 1, but the improvement is not large; the decellularized amniotic membrane material prepared in Comparative Example 2 has not only not improved the rate of promoting the proliferation of epidermal cells compared with that in Example 1, but has decreased; this shows that: the macroporous resin elution conditions of the present invention are also very critical; the decellularized amniotic membrane must be soaked in a natural extract prepared by further subjecting the saffron-Ganoderma lucidum organic solvent extract to the above-mentioned macroporous resin elution conditions of the present invention, compared with soaking in the saffron-Ganoderma lucidum organic solvent extract as a natural extract, in order to further significantly improve the cell proliferation promoting effect of the prepared decellularized amniotic membrane material; however, soaking in a natural extract prepared by further subjecting the saffron-Ganoderma lucidum organic solvent extract to other macroporous resin elution conditions cannot further significantly improve the cell proliferation promoting effect of the prepared decellularized amniotic membrane material.
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410536649.9ACN118416309B (en) | 2024-04-30 | 2024-04-30 | Acellular amniotic membrane material, preparation method thereof and application thereof in preparation of ocular surface repair material |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410536649.9ACN118416309B (en) | 2024-04-30 | 2024-04-30 | Acellular amniotic membrane material, preparation method thereof and application thereof in preparation of ocular surface repair material |
| Publication Number | Publication Date |
|---|---|
| CN118416309Atrue CN118416309A (en) | 2024-08-02 |
| CN118416309B CN118416309B (en) | 2024-12-20 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410536649.9AActiveCN118416309B (en) | 2024-04-30 | 2024-04-30 | Acellular amniotic membrane material, preparation method thereof and application thereof in preparation of ocular surface repair material |
| Country | Link |
|---|---|
| CN (1) | CN118416309B (en) |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6333193B1 (en)* | 1998-08-28 | 2001-12-25 | Nipro Corporation | Cell growth accelerator and cell growth method using the same |
| US20030187515A1 (en)* | 2002-03-26 | 2003-10-02 | Hariri Robert J. | Collagen biofabric and methods of preparing and using the collagen biofabric |
| JP2009234940A (en)* | 2008-03-26 | 2009-10-15 | Toyobo Co Ltd | Cosmetic composition containing vegetable extract |
| JP2010132606A (en)* | 2008-12-05 | 2010-06-17 | Noevir Co Ltd | Moisturizing agent, antiaging agent, antioxidation agent, slimming agent, whitening agent, anti-inflammatory agent, immunostimulating agent, external preparation for skin and functional oral administration composition |
| US9295753B1 (en)* | 2012-07-02 | 2016-03-29 | Celso Tello | Amniotic membrane preparation and device for use as a lens or as a dressing for promoting healing |
| US20210254056A1 (en)* | 2017-05-05 | 2021-08-19 | Camp4 Therapeutics Corporation | Identification and targeted modulation of gene signaling networks |
| CN114712562A (en)* | 2022-04-08 | 2022-07-08 | 上海亚朋生物技术有限公司 | A kind of preparation technology of acellular freeze-dried amniotic membrane product |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6333193B1 (en)* | 1998-08-28 | 2001-12-25 | Nipro Corporation | Cell growth accelerator and cell growth method using the same |
| US20030187515A1 (en)* | 2002-03-26 | 2003-10-02 | Hariri Robert J. | Collagen biofabric and methods of preparing and using the collagen biofabric |
| JP2009234940A (en)* | 2008-03-26 | 2009-10-15 | Toyobo Co Ltd | Cosmetic composition containing vegetable extract |
| JP2010132606A (en)* | 2008-12-05 | 2010-06-17 | Noevir Co Ltd | Moisturizing agent, antiaging agent, antioxidation agent, slimming agent, whitening agent, anti-inflammatory agent, immunostimulating agent, external preparation for skin and functional oral administration composition |
| US9295753B1 (en)* | 2012-07-02 | 2016-03-29 | Celso Tello | Amniotic membrane preparation and device for use as a lens or as a dressing for promoting healing |
| US20210254056A1 (en)* | 2017-05-05 | 2021-08-19 | Camp4 Therapeutics Corporation | Identification and targeted modulation of gene signaling networks |
| CN114712562A (en)* | 2022-04-08 | 2022-07-08 | 上海亚朋生物技术有限公司 | A kind of preparation technology of acellular freeze-dried amniotic membrane product |
| Publication number | Publication date |
|---|---|
| CN118416309B (en) | 2024-12-20 |
| Publication | Publication Date | Title |
|---|---|---|
| CN102824377B (en) | Method for extracting functional ingredients from lucid ganoderma sporocarp | |
| CN106883304A (en) | Heterogeneity polysaccharide is comprehensively prepared and purification process in a kind of Hericium erinaceus | |
| CN101979654A (en) | A method for extracting collagen from fish skin | |
| CN105061633B (en) | A kind of method that fucoidin is produced using sargassum | |
| CN103039831A (en) | Sophora flower health-care rice cake and preparation method thereof | |
| CN106265459A (en) | A kind of Herba Dendrobii promotes skin regeneration combination anti-senile preparation | |
| CN102351826A (en) | Extraction method of ginkgo leaf flavonoids based on steam explosion | |
| CN107488627B (en) | Biological gel for treating refractory skin damage and application thereof | |
| CN118416309A (en) | A decellularized amniotic membrane material and its preparation method and its application in preparing ocular surface repair material | |
| CN107793492A (en) | A kind of extracting method of fructus cannabis polysaccharide | |
| CN103120302A (en) | Making method of sea cucumber medicinal liquor | |
| DE69532878T2 (en) | A process for purifying viral hepatitis B surface antigen containing a preS2 peptide | |
| CN104231100A (en) | A kind of extraction method of versicolor polysaccharide | |
| CN105412167B (en) | Method for extracting and separating ginkgetin and bilobalide from folium Ginkgo | |
| CN103054911B (en) | Crocodile antineoplastic preparation and production method thereof | |
| TW201740964A (en) | Method for manufacturing fungal pharmaceutical composition | |
| CN106866812B (en) | A method for extracting multiple urinary proteins from women's urine | |
| CN86104409A (en) | The preparation method of total gypenosides | |
| CN104497116A (en) | Method for extracting abelmoschus esculentus polysaccharide peptide from abelmoschus esculentus | |
| CN118416308A (en) | A dry biological amniotic membrane material and its preparation method and application as an ophthalmic material | |
| CN114190362A (en) | Placenta preserving fluid and placenta preserving method | |
| CN113637093A (en) | Method for extracting and separating polysaccharide and saponin from asparagus leftovers | |
| CN106832031A (en) | The preparation method of plantago asiatica linne polyoses extract | |
| CN104961744B (en) | A method of extracting lycorine from lycoris plants | |
| CN103142718A (en) | Liquid medicine for treating freezing injury |
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right | Denomination of invention:A decellularized amniotic membrane material and its preparation method, and its application in the preparation of ocular surface repair materials Granted publication date:20241220 Pledgee:CITIC Bank Co.,Ltd. Guangzhou Branch Pledgor:GUANGZHOU YUEQING REGENERATION MEDICINE TECHNOLOGY Co.,Ltd. Registration number:Y2025980007352 |