胰腺内皮细胞共培养对C3A细胞功能变化的影响

Objective To investigate the functional changes of C3A cells co-cultured with pancreatic endothelial cells. Methods C3A cells and pancreatic endothelial cells lwere cultivated directly and indirectly for 7 days by the density ratio of 10: 1. Meanwhile, the blank control was established.The growth and morphological characteristics of experimental groups were observed. The out leakage level of AST, ALT and the synthesis level of albumin and the diazepam metabolism level were determined by automatic biochemistry analyzer, radioimmunoassay and enzyme linked immunosorbent assay. Results Direct co-cultivation of C3A cells and pancreatic endothelial cells could reduce the out leakage of ALT and AST in the 5th and 7th d. The capacity of albumin synthesis could reach 12. 977μg/ml on the 7th d and the that of diazepam metabolism could also be improved. Indirect co-cultivation of C3A cells and pancreatic endothelial cells could significantly raise the albumin synthesis to 7. 380 μg/ml and 10. 773 μg/ml on the 5th and 7th d, respectively. Conclusion Co-cultivation of C3A cells and pancreatic endothelial cells can significantly improve the basic biological function of C3A cells.     

大鼠肾上腺细胞经胶原凝胶三维培养后进行同种移植的实验研究

Objective To investigate a novel method of adrenocotical cells transplantation. Methods Adrenal glands of neonate rats were dissociated into adrenal cortical cells. Cells were cultured in self-made collagen type Ⅰ gel for one week, and then transplanted under the renal capsule of bilateral adrenalectomy mate rats. Blood samples were collected per week after surgery. Animals were sacrificed at the 8th week, and histological characteristics of the allografts, proliferation of transplanted cells, CYP11B1 and CYP11B2 expression as well as plasma aldosterone and corticosterone were observed. Results There were totally 15 rats receiving collagen gel transplantation, and 12 survived 8 weeks post-operation. The plasma cortocosterone level in collagen gel transplantation group was significantly higher than in adrenocortical cell transplantation group, and reached the normal level from 6th to 8th week, but the change in plasma aldosterone level in collagen gel transplantation group was similar to that of adrenocortical cell transplantation group. The adrenocortical cells cultured in gel grew well, and had a high proliferation rate. 95% of them were fasciculata cells which expressed CYP11B1, and the rest were glomerulosa cells which expressed CYP11B2. No inflammatory cell infiltration was observed. Conclusion The proliferation and function of adrenocortical cells could be promoted when they were cultured in collagen gel.     

大鼠肾上腺细胞经胶原凝胶三维培养后进行同种移植的实验研究

Objective To investigate a novel method of adrenocotical cells transplantation. Methods Adrenal glands of neonate rats were dissociated into adrenal cortical cells. Cells were cultured in self-made collagen type Ⅰ gel for one week, and then transplanted under the renal capsule of bilateral adrenalectomy mate rats. Blood samples were collected per week after surgery. Animals were sacrificed at the 8th week, and histological characteristics of the allografts, proliferation of transplanted cells, CYP11B1 and CYP11B2 expression as well as plasma aldosterone and corticosterone were observed. Results There were totally 15 rats receiving collagen gel transplantation, and 12 survived 8 weeks post-operation. The plasma cortocosterone level in collagen gel transplantation group was significantly higher than in adrenocortical cell transplantation group, and reached the normal level from 6th to 8th week, but the change in plasma aldosterone level in collagen gel transplantation group was similar to that of adrenocortical cell transplantation group. The adrenocortical cells cultured in gel grew well, and had a high proliferation rate. 95% of them were fasciculata cells which expressed CYP11B1, and the rest were glomerulosa cells which expressed CYP11B2. No inflammatory cell infiltration was observed. Conclusion The proliferation and function of adrenocortical cells could be promoted when they were cultured in collagen gel.     

大鼠肾上腺细胞经胶原凝胶三维培养后进行同种移植的实验研究

Objective To investigate a novel method of adrenocotical cells transplantation. Methods Adrenal glands of neonate rats were dissociated into adrenal cortical cells. Cells were cultured in self-made collagen type Ⅰ gel for one week, and then transplanted under the renal capsule of bilateral adrenalectomy mate rats. Blood samples were collected per week after surgery. Animals were sacrificed at the 8th week, and histological characteristics of the allografts, proliferation of transplanted cells, CYP11B1 and CYP11B2 expression as well as plasma aldosterone and corticosterone were observed. Results There were totally 15 rats receiving collagen gel transplantation, and 12 survived 8 weeks post-operation. The plasma cortocosterone level in collagen gel transplantation group was significantly higher than in adrenocortical cell transplantation group, and reached the normal level from 6th to 8th week, but the change in plasma aldosterone level in collagen gel transplantation group was similar to that of adrenocortical cell transplantation group. The adrenocortical cells cultured in gel grew well, and had a high proliferation rate. 95% of them were fasciculata cells which expressed CYP11B1, and the rest were glomerulosa cells which expressed CYP11B2. No inflammatory cell infiltration was observed. Conclusion The proliferation and function of adrenocortical cells could be promoted when they were cultured in collagen gel.     

大鼠肾上腺细胞经胶原凝胶三维培养后进行同种移植的实验研究

Objective To investigate a novel method of adrenocotical cells transplantation. Methods Adrenal glands of neonate rats were dissociated into adrenal cortical cells. Cells were cultured in self-made collagen type Ⅰ gel for one week, and then transplanted under the renal capsule of bilateral adrenalectomy mate rats. Blood samples were collected per week after surgery. Animals were sacrificed at the 8th week, and histological characteristics of the allografts, proliferation of transplanted cells, CYP11B1 and CYP11B2 expression as well as plasma aldosterone and corticosterone were observed. Results There were totally 15 rats receiving collagen gel transplantation, and 12 survived 8 weeks post-operation. The plasma cortocosterone level in collagen gel transplantation group was significantly higher than in adrenocortical cell transplantation group, and reached the normal level from 6th to 8th week, but the change in plasma aldosterone level in collagen gel transplantation group was similar to that of adrenocortical cell transplantation group. The adrenocortical cells cultured in gel grew well, and had a high proliferation rate. 95% of them were fasciculata cells which expressed CYP11B1, and the rest were glomerulosa cells which expressed CYP11B2. No inflammatory cell infiltration was observed. Conclusion The proliferation and function of adrenocortical cells could be promoted when they were cultured in collagen gel.     

The relationship between CD4-CD8-T cells in the peripheral blood of patients with pancreatic carcinoma and IL-4,IFN-γ levels

Objective To investigate the relationship between CD4-CD8-T cells ratio and IL-4, IFN-γ levels in the peripheral blood of patients with pancreatic carcinoma. Methods Floweytometer was used to analyze the CD4-CD8-T cells ratio in the peripheral blood of patients with pancreatic carcinoma and the IL-4 ,IFN-γ levels were detected by ELISA. Results The ratio of CD4-CD8-T cell in CD3+ T cell from 25 pancreatic carcinoma patients was(4.2 ± 1.7) %, the ratio of CD4-CD8-T cell in CD3+T cell from 45 healthy person was(6.3 ± 2.6) %, there was significant deviation between the two groups(P ＜ 0. 01). The IL-4 level of 25 pancreatic carcinoma patients was (86.3 ± 23.3) fg/L,the IL-4 level of 45 healthy person was (56.2 ± 9.2) fg/L,there was significant deviation between the two groups(P ＜0.01). The IFN-γ level of 25 pancreatic carcinoma patients was (16.4±4.8) fg/L before operation, the IFN-γ level of 45 healthy person was (27.4±3.8) fg/L,there was significant deviation between the two groups(P ＜0.01). The ratio of CD4-CD8-T cell in pancreatic carcinoma patient after operation was higher than before operation. It could be found negative correlation between CD4-CD8-T cells ratio and IL-4 level in pancreatic carcinoma patient,it could also be found positive correlation between CD4-CD8-T cells ratio and IFN-γ level in pancreatic carcinoma patient. In pancreatic carcinoma patient,the CD4-CD8-T cells ratio and IL-4 level was significant associated with clinical stage (P ＜ 0.05), but no relationship with hisological differentiation (P＞0.05), it could be found no relationship between IFN-γ level and clinical stage, hisoiogieal differentiation (P ＞ 0.05). Conclusion The CD4±CD8±T cells ratio in the peripheral blood of patients is decreased,it may be participate in the carcinogenesis and development of pancreatic carcinoma by influence the IFN-γ levels.     

The relationship between CD4-CD8-T cells in the peripheral blood of patients with pancreatic carcinoma and IL-4,IFN-γ levels

Objective To investigate the relationship between CD4-CD8-T cells ratio and IL-4, IFN-γ levels in the peripheral blood of patients with pancreatic carcinoma. Methods Floweytometer was used to analyze the CD4-CD8-T cells ratio in the peripheral blood of patients with pancreatic carcinoma and the IL-4 ,IFN-γ levels were detected by ELISA. Results The ratio of CD4-CD8-T cell in CD3+ T cell from 25 pancreatic carcinoma patients was(4.2 ± 1.7) %, the ratio of CD4-CD8-T cell in CD3+T cell from 45 healthy person was(6.3 ± 2.6) %, there was significant deviation between the two groups(P ＜ 0. 01). The IL-4 level of 25 pancreatic carcinoma patients was (86.3 ± 23.3) fg/L,the IL-4 level of 45 healthy person was (56.2 ± 9.2) fg/L,there was significant deviation between the two groups(P ＜0.01). The IFN-γ level of 25 pancreatic carcinoma patients was (16.4±4.8) fg/L before operation, the IFN-γ level of 45 healthy person was (27.4±3.8) fg/L,there was significant deviation between the two groups(P ＜0.01). The ratio of CD4-CD8-T cell in pancreatic carcinoma patient after operation was higher than before operation. It could be found negative correlation between CD4-CD8-T cells ratio and IL-4 level in pancreatic carcinoma patient,it could also be found positive correlation between CD4-CD8-T cells ratio and IFN-γ level in pancreatic carcinoma patient. In pancreatic carcinoma patient,the CD4-CD8-T cells ratio and IL-4 level was significant associated with clinical stage (P ＜ 0.05), but no relationship with hisological differentiation (P＞0.05), it could be found no relationship between IFN-γ level and clinical stage, hisoiogieal differentiation (P ＞ 0.05). Conclusion The CD4±CD8±T cells ratio in the peripheral blood of patients is decreased,it may be participate in the carcinogenesis and development of pancreatic carcinoma by influence the IFN-γ levels.     

The relationship between CD4-CD8-T cells in the peripheral blood of patients with pancreatic carcinoma and IL-4,IFN-γ levels

Objective To investigate the relationship between CD4-CD8-T cells ratio and IL-4, IFN-γ levels in the peripheral blood of patients with pancreatic carcinoma. Methods Floweytometer was used to analyze the CD4-CD8-T cells ratio in the peripheral blood of patients with pancreatic carcinoma and the IL-4 ,IFN-γ levels were detected by ELISA. Results The ratio of CD4-CD8-T cell in CD3+ T cell from 25 pancreatic carcinoma patients was(4.2 ± 1.7) %, the ratio of CD4-CD8-T cell in CD3+T cell from 45 healthy person was(6.3 ± 2.6) %, there was significant deviation between the two groups(P ＜ 0. 01). The IL-4 level of 25 pancreatic carcinoma patients was (86.3 ± 23.3) fg/L,the IL-4 level of 45 healthy person was (56.2 ± 9.2) fg/L,there was significant deviation between the two groups(P ＜0.01). The IFN-γ level of 25 pancreatic carcinoma patients was (16.4±4.8) fg/L before operation, the IFN-γ level of 45 healthy person was (27.4±3.8) fg/L,there was significant deviation between the two groups(P ＜0.01). The ratio of CD4-CD8-T cell in pancreatic carcinoma patient after operation was higher than before operation. It could be found negative correlation between CD4-CD8-T cells ratio and IL-4 level in pancreatic carcinoma patient,it could also be found positive correlation between CD4-CD8-T cells ratio and IFN-γ level in pancreatic carcinoma patient. In pancreatic carcinoma patient,the CD4-CD8-T cells ratio and IL-4 level was significant associated with clinical stage (P ＜ 0.05), but no relationship with hisological differentiation (P＞0.05), it could be found no relationship between IFN-γ level and clinical stage, hisoiogieal differentiation (P ＞ 0.05). Conclusion The CD4±CD8±T cells ratio in the peripheral blood of patients is decreased,it may be participate in the carcinogenesis and development of pancreatic carcinoma by influence the IFN-γ levels.     

大鼠肾上腺细胞经胶原凝胶三维培养后进行同种移植的实验研究

Objective To investigate a novel method of adrenocotical cells transplantation. Methods Adrenal glands of neonate rats were dissociated into adrenal cortical cells. Cells were cultured in self-made collagen type Ⅰ gel for one week, and then transplanted under the renal capsule of bilateral adrenalectomy mate rats. Blood samples were collected per week after surgery. Animals were sacrificed at the 8th week, and histological characteristics of the allografts, proliferation of transplanted cells, CYP11B1 and CYP11B2 expression as well as plasma aldosterone and corticosterone were observed. Results There were totally 15 rats receiving collagen gel transplantation, and 12 survived 8 weeks post-operation. The plasma cortocosterone level in collagen gel transplantation group was significantly higher than in adrenocortical cell transplantation group, and reached the normal level from 6th to 8th week, but the change in plasma aldosterone level in collagen gel transplantation group was similar to that of adrenocortical cell transplantation group. The adrenocortical cells cultured in gel grew well, and had a high proliferation rate. 95% of them were fasciculata cells which expressed CYP11B1, and the rest were glomerulosa cells which expressed CYP11B2. No inflammatory cell infiltration was observed. Conclusion The proliferation and function of adrenocortical cells could be promoted when they were cultured in collagen gel.     

大鼠肾上腺细胞经胶原凝胶三维培养后进行同种移植的实验研究

Objective To investigate a novel method of adrenocotical cells transplantation. Methods Adrenal glands of neonate rats were dissociated into adrenal cortical cells. Cells were cultured in self-made collagen type Ⅰ gel for one week, and then transplanted under the renal capsule of bilateral adrenalectomy mate rats. Blood samples were collected per week after surgery. Animals were sacrificed at the 8th week, and histological characteristics of the allografts, proliferation of transplanted cells, CYP11B1 and CYP11B2 expression as well as plasma aldosterone and corticosterone were observed. Results There were totally 15 rats receiving collagen gel transplantation, and 12 survived 8 weeks post-operation. The plasma cortocosterone level in collagen gel transplantation group was significantly higher than in adrenocortical cell transplantation group, and reached the normal level from 6th to 8th week, but the change in plasma aldosterone level in collagen gel transplantation group was similar to that of adrenocortical cell transplantation group. The adrenocortical cells cultured in gel grew well, and had a high proliferation rate. 95% of them were fasciculata cells which expressed CYP11B1, and the rest were glomerulosa cells which expressed CYP11B2. No inflammatory cell infiltration was observed. Conclusion The proliferation and function of adrenocortical cells could be promoted when they were cultured in collagen gel.     

胰腺内皮细胞共培养对C3A细胞功能变化的影响

目的 探讨与胰腺内皮细胞共培养时C3A细胞功能变化.方法 选择C3A细胞与胰腺内皮细胞的密度比例为10:1分别进行直接和间接共培养7 d,同时设立空白对照组,观察各实验组细胞生长和形态特征,分别用杜邦全自动生化分析仪、放射免疫分析法和ELISA法检测培养液中AST、ALT漏出量,白蛋白含量和安定代谢量.结果 C3A细胞与胰腺内皮细胞直接共培养第5、7天能显著降低ALT、AST的漏出量,第7天提高白蛋白合成量最高达12.977μg/ml和提高安定代谢能力;C3A细胞与胰腺内皮细胞间接共培养在第5、7天也能显著提高白蛋白合成量到7.380μg/ml、10.773μg/ml.结论 与胰腺内皮细胞直接与间接共培养均能明显改善C3A细胞的功能.     

Culture of C3A cells in alginate beads for fluidized bed bioartificial liver

Extracorporeal bioartificial liver has been designed to sustain the detoxification and synthetic function of the failed liver in patients suffering from acute liver failure until the time of liver allotransplantation or regeneration of their own. A fluidized bed, bioartificial liver improves the mass transfer velocity between the medium and the hepatocytes. Detoxification functions of the liver could be replaced by completely artificial systems, but the synthetic functions of hepatocytes may be obtained only by metabolically active cells. The aim of our study was to investigate the influence of C3A cell culture in alginate beads on synthetic function in a fluidized bed, bioartificial liver. Cells in alginate beads were prepared using an electrostatic droplet generator of our own design using low-viscosity alginate. Beads were cultured for 24 hours then 7 days in static conditions and then 24 hours of fluidization in the bioreactor to assess albumin production. We observed significantly increased albumin production by C3A cells entrapped in alginate beads during static culture. Fluidization increased albumin production compared with static culture. Fluidization performed after 7 days of static culture resulted in a significant increase in albumin synthesis. In conclusion, static culture of alginate beads hosting hepatic cells facilitates restoration of cell function.     

新颖海洋化合物SZ-685C抑制GH3细胞增殖并诱导其凋亡

目的 探讨SZ-685C对大鼠垂体生长激素腺瘤细胞(GH3)系的增殖和凋亡影响. 方法 用胎鼠成纤维细胞(MEF)作为对照,MTT检测0、2.5、5.0、7.5、10.0、12.5、15.0、17.5、20.0和30.0 μmol/LSZ-685C对GH3细胞增殖活性影响和药物的半数抑制浓度(IC50),结晶紫染色观察不同浓度SZ-685C对GH3细胞集群生长的影响;光镜和Hoechst 33342染色观察细胞凋亡的形态学变化,流式细胞术检测细胞凋亡率. 结果 SZ-685C能抑制GH3细胞的增殖,并具有浓度依赖性,IC50为(12.9±0.47)μmol/L,而作为正常对照的MEF在此浓度下,细胞增殖活性没有明显变化;SZ-685C作用GH3细胞48 h后更换完全培养基培养12d,结晶紫染色观察肿瘤细胞的集群生长仍受到明显抑制,表现为克隆数的骤减;光镜和Hoechst 33342染色观察到细胞发生凋亡的形态学证据,流式细胞的检测在不同浓度SZ-685C的作用下,细胞凋亡比例从2.0％上升至36.4％. 结论 SZ-685C能抑制GH3细胞增殖,其增殖抑制作用可能通过促进细胞凋亡的方式,加速了细胞死亡进程,为其可能作为临床新型抗垂体瘤药物提供了初步的实验依据.     

Humanized C3 Mouse: A Novel Accelerated Model of C3 Glomerulopathy

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Spongy polyethersulfone membrane for hepatocyte cultivation: studies on human hepatoma C3A cells

There are different types of membranes used for hepatocyte cultivation. In our studies, spongy polyethersulfone (PES) membranes were examined as a support for hepatic cell cultivation in vitro. The extended surface of the membranes allows to introduce a high cell number especially in three-dimensional gel structure. Scanning electron microscopy analysis indicated that C3A cells used in our experiments grew well on PES membranes forming microvilli characteristic for normal hepatocytes. Analysis of cell viability proved that spongy PES membrane is well tolerated by J774 macrophages and did not stimulate nitric oxide synthesis. Bile canalicular structures were observed in fluorescence microscopy after F-actin staining with tetramethyl rhodamine iso-thiocyanate (TRITC)-phalloidin. The C3A cells showed high affinity to the PES membranes and adhered to almost 90% during the initial 24 h of incubation. Albumin production increased during static culture from the value of 805.2 +/- 284.4 (ng/24 h/initial 10(6) cells) during the first days, to 2017.6 +/- 505.9 (ng/24 h/initial 10(6) cells) after 10 days of culture. In conclusion, the spongy PES membranes can be used as scaffold for hepatocyte cultivation, especially for the creation of three-dimensional environments.     

Demonstration and characterization of C3 receptors on rat glomerular epithelial cells

Receptors for C3 have been demonstrated on the glomerular podocyte in humans. There is conflicting evidence regarding the presence of C3 receptors on rat glomerular cells. Even when shown to be present, the ligand specificity of the receptor has not been determined. Decapsulated rat glomeruli obtained from male Sprague-Dawley rats weighing 50 to 100 g were placed in enriched culture media. On days four to eight, cells of epithelial morphology were observed growing out of glomeruli. Receptors for C3 were detected by rosette formation of sheep erythrocytes (E) coated with antibody (A) and complement (EAC) around the glomerular epithelial cells in culture. The EACs were prepared by incubating antibody-coated sheep erythrocytes with C5-deficient mouse serum or with individual components of complement. Results indicate the presence of two types of C3 receptors on glomerular epithelial cells--CR1 for C3b and CR2 for C3d. The functional roles of these receptors remain to be elucidated.     

抑制核纤层蛋白A/C表达对牵张刺激下成骨细胞增殖及凋亡的影响

目的 观察小干扰RNA技术(siRNA)抑制核纤层蛋白A/C(lamin A/C)表达对牵张刺激下小鼠前成骨细胞MC3T3-E1增殖及凋亡的影响.方法 化学合成3条针对lamin A/C靶点的siRNA序列,脂质体转染MC3T3-E1细胞,实时定量聚合酶链反应(PCR)和Western blot检测lamin A/C mRNA和蛋白变化.细胞转染72 h后行牵张刺激6 h,Hoechst 33258检测DNA含量来反映细胞增殖,流式细胞术检测细胞周期及凋亡.结果 筛选出的siRNA-2显著抑制lamin A/C mRNA和蛋白产物表达(P＜0.01).未转染细胞经牵张刺激,DNA合成随时间推移而增加;细胞G0/G1期比例为65.19%;S期为22.57%,细胞凋亡率为11.49%;转染细胞经牵张刺激后,DNA合成较未转染细胞显著性减少(P＜0.01);G0/G1期比例提高至85.82%;S期降低至11.37%,凋亡率达19.32%(P＜0.01).结论 抑制lamin A/C表达会导致牵张刺激诱导的促增殖作用减弱,细胞周期阻滞于G0/G1期,促进细胞的凋亡.     

C3G overexpression in glomerular epithelial cells during anti-GBM-induced glomerulonephritis

The guanine nucleotide exchange factor C3G, along with the CrkII adaptor protein, mediates GTP activation of the small GTPase proteins Rap1 and R-Ras, facilitating their activation of downstream signaling pathways, which had been found to be important in the pathogenesis of glomerulonephritis. We found that expression of C3G protein was upregulated in glomerular epithelial cells in an experimental model of accelerated anti-GBM antibody-induced glomerulonephritis expression. To determine the consequence of its increased expression, we transfected C3G (using adenoviral constructs) into cultured glomerular epithelial cells and measured the activated forms (i.e., GTP-bound) forms of Rap1 and R-Ras. Activation of Rap1 was not affected by C3G; however, the basal level of GTP-bound R-Ras was decreased. Further, C3G over-expression enhanced the activation of R-Ras in response to endothelin. Overexpression of C3G also led to a significant reduction in glomerular epithelial cell spreading and decreased the cells' E-cadherin expression and augmented their migration. We found that C3G was overexpressed in accelerated anti-GBM antibody-induced glomerulonephritis and suggest that this modulates glomerular epithelial cell morphology and behavior.