摘要
采用肝脏原位灌流法分离大鼠肝细胞 ,以血纤维蛋白膜作支架 ,在RWVB回转器提供的模拟微重力条件下 ,对肝细胞进行三维培养。肝细胞经连续培养 2 8d后 ,细胞仍呈球状 ,并获得了三维培养条件下大鼠肝细胞团 (约为 2 0 0～ 30 0个 /细胞团 )。三维培养的肝细胞在培养期间具有持续分泌ALB和TBA的功能 ,而单层培养的肝细胞仅在接种后 18d内具有一定分泌功能 ,之后细胞逐渐衰老死亡。三维培养的肝细胞培养至 2 8d时仍可检测到G6PD、PFK、PGM三种糖代谢关键酶基因的转录 ,而单层培养的肝细胞在接种后第 6d就检测不到PFK、PGM的转录。结果表明 ,模拟微重力条件下三维培养的肝细胞在培养期间不仅能维持正常细胞形态 ,而且具有稳定的分泌功能和糖代谢功能 ,而单层培养的肝细胞分泌功能显著下降 ,糖代谢功能出现异常。
day old Sprague Dawley rats were selected for the isolation of hepatocytes by the method of in situ perfusion. A needle was inserted into the foramen occipital magnum of each rats and cerebral paralysis was induced by brain lesion. The abdominal cavity was opened to expose the liver. Collagenase (type Ⅰ) at the concentration of 0 5 g/L was continuously perfused into the postcaval vein for 10 minutes. The liver was removed and placed in a Petri dish and hepatocytes released after the capsula were torn to pieces with forceps. The hepatocytes were collected and washed three times with RPMI 1640 medium.\; In order to get scaffolds for hepatocyte culture, blood from adult rats was collected by removing their eyeball and placing these in a Petri dish. After coagulation, resultant blood clots were rinsed thoroughly with 0 9% NaCl. The fiblin from the blood was lyophilized and cut into 1 mm 3 pieces. After sterilization by exposure to 60 Co , the fiblin was stored at 4 ℃. To create a monolayer culture, the fiblin was spread over six well plates, and the medium of RPMI 1640 supplemented with 15% fetal sera was added into the plates. Hepatocytes were inoculated onto the fiblin at a concentration of 2×10 5cells/ml. The plates were then put into the incubator and cultured at 37℃ and the hepatocytes observed daily under a microscope. On days 2, 3, 5, 7, 9, 11, 14 and 18 of culture, the medium was collected and the concentrations of albumin and bile acid in medium it tested with an Automatic Chemical Analyzer (Beckman CX 7). In the three dimension culture, the simulated microgravity condition was created with a Rotating wall Vessel Bioreactor (RWVB). After the hepatocytes and the fiblin were mixed and pre cultured in RPMI 1640 for 30 minutes, the mixture was transferred into RWVB with the final concentration of hepatocytes being 2×10 5cells/ml. The rotating speed of the vessel was set at 15 rpm in the first week of culture, 20 rpm in the second week and 30 rpm for the final stage. The hepatocytes were observed with a microscope. On days 2, 3, 5, 7, 9, 11, 14, 18, 20, 24 and, 28 of culture, the medium was collected and the concentrations of albumin and bile acid in medium tested in the same way as for the monolayer culture. To compare the transcript expression of the genes of glucose 6 phosphate dehydrogenase (G6PDH), phospho fructokinase (PFK), and phosphoglucomutase (PGM) of hepatocytes cultured in both the monolayer and three dimensional condition, RNA was extracted from hepatocytes cultured on day 2, 4, and 6 in the monolayer condition and from hepatocytes cultured on day 28 in the three dimension condition. RNA from hepatocytes isolated from fresh liver tissue was also extracted as a control. The mRNA of G6PDH, PFK and PGM in all the above samples was detected by nested RT PCR. The results showed that rat hepatocytes cultured under simulated microgravity conditions began to attach to the scaffolds in RWVB after 12 hours of inoculation. Forty eight hours after inoculation, most of the cells had aggregated on the edge of scaffolds and small clumps of cells had emerged. After one week of inoculation, the hepatocytes clumps were connected with each other through an extra cellular matrix which made the clumps grow larger. After 28 days of culture, hepatocytes clumps with diameters of 500 to 600 micrometers each containing 200 to 300 cells had formed. Compared to hepatocytes cultured under monolayer conditions, which were markedly stretched and deformed, hepatocytes cultured under simulated microgravity remained spherical throughout the entire period of 28 days of culture. Moreover, hepatocytes cultured under simulated microgravity could continuously secrete albumin and bile acid throughout all 28 days of culture, while those under monolayer conditions could do so only within the first 18 days of culture. The transcript expression of the genes of glucose 6 phosphate dehydrogenase (G6PDH), phospho fructokinase (PFK), and phosphoglucomutase (PGM) could be detected by nested
出处
《动物学报》
SCIE
CAS
CSCD
北大核心
2002年第6期770-776,共7页
ACTA ZOOLOGICA SINICA
基金
国家 8 63 2项目资助项目 (863 2 7 2 17)~~
