CN100460505C - Hepatocyte Culture Method Using Polylactic Acid-O-Carboxymethyl Chitosan Nanoparticles - Google Patents

Abstract

The invention discloses a poly-lactic acid-0-carboxymethyl chitosan nanometer particle hepatocyte culturing method. It mixes poly lactic acid-0-carboxymethyl chitosan nanometer particle and hepatocyte to do inoculated culture in culturing process. The invention can effectively accelerate hepatocyte growth, increase its energy, and will play an importance role in hepatocyte transplantation.

Description

Translated fromChinese

用聚乳酸—O—羧甲基壳聚糖纳米粒子的肝细胞培养方法Hepatocyte Culture Method Using Polylactic Acid-O-Carboxymethyl Chitosan Nanoparticles

技术领域：Technical field:

本发明涉及一种肝细胞的培养方法。The invention relates to a method for culturing hepatocytes.

背景技术：Background technique:

急性肝衰竭(Acute liver failure，ALF)是各种肝脏疾病特别是病毒性肝炎的主要死因之一。肝细胞移植可以起到一定的肝功能支持作用。由于其具有操作简单、对机体生理环境干扰小、供体来源广泛等优点，已引起了众多研究者的广泛关注。然而，如何提高移植肝细胞的活性和降低免疫排斥反应是急需解决的两大问题。Acute liver failure (ALF) is one of the leading causes of death in various liver diseases, especially viral hepatitis. Liver cell transplantation can play a certain role in supporting liver function. Because of its advantages of simple operation, less interference to the physiological environment of the body, and a wide range of donor sources, it has attracted extensive attention from many researchers. However, how to improve the activity of transplanted hepatocytes and reduce immune rejection are two problems that need to be solved urgently.

发明内容：Invention content:

本发明的目的在于提供一种可有效促进肝细胞生长，提高肝细胞活力的用聚乳酸—O—羧甲基壳聚糖纳米粒子的肝细胞培养方法。The object of the present invention is to provide a method for culturing liver cells with polylactic acid-O-carboxymethyl chitosan nanoparticles that can effectively promote the growth of liver cells and improve the vitality of liver cells.

本发明的技术解决方案是：Technical solution of the present invention is:

一种用聚乳酸—O—羧甲基壳聚糖纳米粒子的肝细胞培养方法，其特征是：在进行肝细胞培养时，将聚乳酸—O—羧甲基壳聚糖纳米粒子与肝细胞混合接种培养。A method for culturing liver cells using polylactic acid-O-carboxymethyl chitosan nanoparticles, which is characterized in that: when culturing liver cells, polylactic acid-O-carboxymethyl chitosan nanoparticles and liver cells Mixed inoculation culture.

培养时温度为35～38℃，并且在5％浓度的CO₂条件下培养，培养12小时内，每30分钟震动一次，每次持续5分钟。The temperature during cultivation is 35-38° C., and the cultivation is carried out under the condition of 5% CO₂ . Within 12 hours of cultivation, shake once every 30 minutes and last for 5 minutes each time.

培养时温度为37℃。The temperature during cultivation was 37°C.

肝细胞与聚乳酸—O—羧甲基壳聚糖纳米粒子的用量关系为：肝细胞5×10⁵个/ml与40μg/ml的聚乳酸—O—羧甲基壳聚糖纳米粒子相混合接种。The dosage relationship between liver cells and polylactic acid-O-carboxymethyl chitosan nanoparticles is as follows: 5×¹⁰⁵ liver cells/ml mixed with 40 μg/ml polylactic acid-O-carboxymethyl chitosan nanoparticles Inoculate.

本发明可有效促进肝细胞生长，提高肝细胞活力，必定为肝细胞移植术的发展起到巨大的推动作用。实验证明，与普通肝细胞培养相比较，本发明培养的肝细胞形成球形聚集体的时间明显缩短，球形聚集体的比例明显增加，细胞形态好，培养后24h、第3天和第5天，ALB含量增高。培养后第3天和第5天，纳米培养组ALT含量低于普通培养组(P<0.05)。两组培养第3天后，ALT含量逐渐降低(P<0.05)。表明新制备的肝细胞悬液由于受到胶原酶和其它因素的影响，肝细胞膜受到不同程度的破坏，而经培养后肝细胞膜重新修复完整；纳米材料更有助于肝细胞膜的修复。The invention can effectively promote the growth of liver cells, improve the vitality of liver cells, and will definitely play a huge role in promoting the development of liver cell transplantation. Experiments have proved that compared with ordinary hepatocyte culture, the time for the hepatocytes cultured in the present invention to form spherical aggregates is significantly shortened, the proportion of spherical aggregates is significantly increased, and the cell shape is good. After 24 hours, 3 days and 5 days after culture, ALB content increased. On the 3rd and 5th day after culture, the ALT content of the nano-culture group was lower than that of the normal culture group (P<0.05). After the third day of culture in both groups, the ALT content gradually decreased (P<0.05). It indicated that the newly prepared hepatic cell suspension was affected by collagenase and other factors, and the hepatic cell membrane was damaged to varying degrees, and the hepatic cell membrane was completely restored after culture; nanomaterials were more helpful to the repair of the hepatic cell membrane.

下面结合实施例对本发明作进一步说明。The present invention will be further described below in conjunction with embodiment.

具体实施方式；Detailed ways;

聚乳酸—O—羧甲基壳聚糖(PLA-O-CMC)纳米粒子的制备：Preparation of polylactic acid-O-carboxymethyl chitosan (PLA-O-CMC) nanoparticles:

将10ml浓度为0.03％的PLA二氯甲烷溶液与10ml浓度为0.15％的O-CMC溶液在超声情况下充分反应(约25min左右)，待其中的有机溶剂发完全后得到乳光较重的均匀悬浊液。将此悬浊液先以2000rpm低速离心5min，取低速离心后所得的上清液以14000rpm高速离心10min，得到部分白色沉淀，吸去部分上清液，将沉淀冷冻抽干取出并置于紫外灯下照射12h，消毒后备用。10ml of 0.03% PLA dichloromethane solution and 10ml of 0.15% O-CMC solution were fully reacted under ultrasonic conditions (about 25min). Suspension. Centrifuge the suspension at a low speed of 2000rpm for 5min, take the supernatant obtained after centrifugation at a low speed and centrifuge at a high speed of 14000rpm for 10min to obtain a part of the white precipitate, absorb part of the supernatant, freeze the precipitate and take it out and put it in a UV lamp Irradiate for 12 hours, and use after disinfection.

猪肝细胞的分离和培养：Isolation and culture of porcine hepatocytes:

采用原位胶原酶循环肝灌注法分离猪肝细胞。将分离所得肝细胞进行培养将肝细胞以5×10⁵个/ml与40μg/ml的PLA-O-CMC纳米粒子相混合接种到培养板上(1ml/孔)，置于37℃、浓度为5％的CO₂条件下静置培养，培养12小时(h)内每30分钟(min)震动一次，每次持续5分钟，以促进纳米材料与肝细胞的结合且有利于细胞集聚形成球形体。培养12h后，细胞贴附在纳米材料上。培养48h后，形成细胞粘附聚集的球形体，提高了细胞密度。培养24h，大部分肝细胞仍保持圆球状，部分肝细胞向多边形转变，多数肝细胞相互黏连，成束状生长。约培养48h后，大部分肝细胞重建细胞极性，呈现较典型、均匀的多边形形态特征。Porcine hepatocytes were isolated by in situ collagenase circulating liver perfusion. The isolated hepatocytes were cultured. The hepatocytes were mixed with 5×10⁵ cells/ml and 40 μg/ml PLA-O-CMC nanoparticles and seeded on a culture plate (1 ml/well), placed at 37° C. at a concentration of Under the condition of 5% CO₂ , culture statically, shake once every 30 minutes (min) within 12 hours (h), and last for 5 minutes each time, so as to promote the combination of nanomaterials and liver cells and facilitate the aggregation of cells to form spheroids . After culturing for 12 hours, the cells were attached to the nanomaterials. After 48 hours of culture, spheroids of cell adhesion aggregation were formed, which increased the cell density. After culturing for 24 hours, most of the hepatocytes remained spherical, some of the hepatocytes transformed into polygons, and most of the hepatocytes adhered to each other and grew in bundles. After culturing for about 48 hours, most of the hepatocytes reestablished their cell polarity and presented a more typical and uniform polygonal shape.

为了更好地分析本发明的效果，同时建立了普通培养组对肝细胞进行培养：将肝细胞以5×10⁵个/ml的浓度接种到培养板上(1ml/孔)，置于37℃、浓度为5％的CO₂条件下静置培养，培养12h内，每30min摇动1次，每次持续5min，以利于细胞集聚形成球形体。In order to better analyze the effect of the present invention, a common culture group has been set up to cultivate the hepatocytes simultaneously: the hepatocytes are inoculated onto the culture plate (1ml/well) at a concentration of 5×10⁵ /ml, placed at 37° C. , The concentration was 5% CO₂ and cultured statically. Within 12 hours of culture, shake once every 30 minutes, and each time lasted 5 minutes, so as to facilitate the aggregation of cells to form spheroids.

普通培养的肝细胞，接种后很快沉于培养板底并贴壁生长，其形态立即由刚分离的圆球形向单层扁平多边形伸展，48～72h后细胞进一步变薄，逐渐失去多边形特征，肝细胞连接成片生长。Ordinary cultured hepatocytes sink to the bottom of the culture plate soon after inoculation and grow on the wall, and their shape immediately extends from a newly separated spherical shape to a single-layer flat polygon. After 48-72 hours, the cells become thinner and gradually lose their polygonal characteristics. Hepatocytes grow in contiguous sheets.

下面表中将用本发明方法的培养组称为“纳米材料培养组”肝细胞功能变化：In the table below, the cultured group using the method of the present invention is called "nanomaterial cultured group" for changes in liver cell function:

两组肝细胞培养上清液中白蛋白(ALB)含量比较(表1)Comparison of albumin (ALB) content in the culture supernatant of hepatocytes between the two groups (Table 1)

       表1 两组肝细胞培养上清液中ALB含量比较(x±s)Table 1 Comparison of ALB content in the culture supernatant of hepatocytes between the two groups (x±s)

经统计学处理，培养后24h、第3天(d)和第5d，纳米细胞培养组ALB含量高于普通培养组(P<0.05)。培养24h后，普通培养组ALB低于培养后12h值(P<0.05)；纳米培养组ALB高于培养后12h值，培养7d后下降。After statistical analysis, 24h, 3rd day (d) and 5th day after culture, the ALB content of the nano-cell culture group was higher than that of the normal culture group (P<0.05). After 24 hours of culture, the ALB of normal culture group was lower than that of 12 hours after culture (P<0.05); the ALB of nano culture group was higher than that of 12 hours after culture, and decreased after 7 days of culture.

两组肝细胞培养上清液中丙氨酸转氨酶(ALT)含量比较(表2)Comparison of alanine aminotransferase (ALT) content in the culture supernatant of the two groups of hepatocytes (Table 2)

  表2 两组肝细胞培养上清液中ALT含量比较(x±s)Table 2 Comparison of ALT content in the supernatant of hepatocyte culture between the two groups (x±s)

经统计学处理，培养后第3d和第5d，纳米材料培养组ALT含量低于普通培养组(P<0.05)。两组培养第3d后，ALT含量逐渐降低(P<0.05)。After statistical analysis, on the 3rd and 5th day after culture, the ALT content of the nanomaterial culture group was lower than that of the normal culture group (P<0.05). After the 3rd day of culture in the two groups, the ALT content gradually decreased (P<0.05).

两组肝细胞培养上清液中尿素氮(BUN)含量比较(表3)Comparison of urea nitrogen (BUN) content in the culture supernatant of liver cells between the two groups (Table 3)

     表3 两组肝细胞培养上清液中BUN含量比较(x±s)Table 3 Comparison of BUN content in the supernatant of hepatocyte culture between the two groups (x±s)

经统计学处理，培养后第3-7天BUN含量有所增高，但各组之间及各时段之间相比差异无显著性(P>0.05)。After statistical processing, the BUN content increased on the 3rd to 7th day after culture, but there was no significant difference between each group and each time period (P>0.05).

Claims (2)

1. liver cell culture method with poly(lactic acid)-O-carboxymethyl chitosan nanoparticles, it is characterized in that: when carrying out liver cell culture, poly(lactic acid)-O-carboxymethyl chitosan nanoparticles and liver cell combined inoculation are cultivated, and temperature is 35～38 ℃ during cultivation, and at the CO of 5% concentration₂Cultivate under the condition, cultivate in 12 hours, vibrations in per 30 minutes once continue 5 minutes at every turn, liver cell and poly(lactic acid)-O-carboxymethyl chitosan nanoparticles with magnitude relation be: liver cell 5 * 10⁵Poly(lactic acid)-O-carboxymethyl chitosan nanoparticles combined inoculation mutually of individual/ml and 40 μ g/ml.

2. the liver cell culture method with poly(lactic acid)-O-carboxymethyl chitosan nanoparticles according to claim 1, it is characterized in that: temperature is 37 ℃ during cultivation.