【Abstract】Objective The effects of tumor infiltrating lymphocytes (TIL) on cellular immunologic function of patients with breast cancer were studied. Methods Twenty five patients with breast cancer were treated by the TIL that were isolated from tissue of tumor. T cell subgroups and natural killer cell (NK cell) activity of peripheral blood, the levels of serum soluble interleukin-2 receptor (sIL-2R) were assayed before and after treatment. Results CD3, CD4, CD4/CD8 and NK cell activity were ascended obviously, and CD8, sIL-2R were descended obviously after the treatment of TIL. Conclusion TIL can enhance the cellular immunologic function of patients with breast cancer.
31 case of advanced primary liver cancer were treated by using IL-2 and LAK cells in which 15 cases were combined with surgery (group A) and 16 cases were combined with chemotherpy (Group B). 7～14 months follow-up showed: In group A there was no recurence and metastasis, and the cell-mediated immunity was obviously improved. In group B, the average life time was more than 5.84 months, the tumor average diameter dicreased in 10 cases ,and the cee-mediated immunity was also improved. The role of immunotherapy combined with surgery or chemotherapy was discussed.
Abstract: Objective To investigate the effect of cytokineinduced killer (CIK) cells immunotherapy on immunity function of non-small cell lung cancer (NSCLC) patients after operation. Methods Fifty patients with histological or cytological diagnosis of NSCLC on Ⅰstage,Ⅱstage andⅢa stage of tumor, nodes, metastasisclassification were randomly divided CIK cells therapy group and conventional therapy group, 25 cases each group. The immunity function of patients with NSCLC, including the levels of CD3+, CD4+ T cells, ratio of CD4+/CD8+, natural killer(NK) cells, and the levels of Th1/Th2 cytokine were detected before treatment, and the 2nd, 4th, 8th week after treatment. Results The levels of CD3+, CD4+ T cells, NK cells, ratio of CD4+/CD8+, interleukin-2(IL-2), interferon-γ(INF-γ) in CIK cells therapy group at the 2nd week after treatment were more higher than those before treatment (Plt;0.01), their levels reached the peak at 4th week, from then on, it began to decrease. Meanwhile, the levels of Th2 of CIK cells therapy group began to decrease at the 2nd week after treatment, a low ebb at 4th week. At the 2nd, 4th and 8th week,the levels of CD3+,CD4+ T cells, ratio of CD4+/CD8+, NK cells,IL-2, INF-γ, interleukin-4(IL-4), interleukin-10(IL-10) of CIK cells therapy group compared with those inconventional therapy group,there were statistical significance difference［(Plt;0.05),at 4th week after treatment, CD3+ 70.2%±9.1% vs.46.3%±5.8%; CD4+40.2%±7.1% vs.22.9%±4.5%; CD4+/CD8+ 1.82±0.43 vs. 1.09±0.34; NK 15.7%±5.4% vs.10.5%±2.5%; IL-2 34.8±11.7 ng/L vs. 19.8±12.1 ng/L; INF-γ63.7±23.3 ng/Lvs. 30.8±10.6 ng/L; IL-4 10.2±8.6 ng/L vs. 25.8±6.3 ng/L; IL-10 10.6±3.4 ng/L vs. 21.4±8.6 ng/L］. Conclusion The results indicate that CIK cells immunotherapy can enhance the immune function of NSCLC patients after operation.
Objective To observe the effects of bevacizumab on aquaporin 4 (AQP4) expression in human retinal Muuml;ller cells in vitro under hypoxia. To explored the mechanism of treating retinal edema with bevacizumab. Methods Human Muuml;ller cells were cultured using the enzymatic digestion method. Transmission electron microscopic analysis and immunofluorescence staining identified Muuml;ller cells. With semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), the expression of AQP4 mRNA and vascular endothelial growth factor （VEGF） mRNA in Muuml;ller cells cultured under different concentration of COCl2 for different hours were observed. The expression of AQP4 mRNA in Muuml;ller cells cultured using CoCl2 precultured with 200 mu;g/ml bevacizumab was measured. Results More than 95% of primary cells showed positive reaction to glial fibrillary acidic protein, glutamine synthetase, vimentin and alpha;-smooth muscle actin with immunofluorescence staining. Characteristic 8-10 nm intracellular filaments could be seen in the cytoplasm viewed with transmission electron microscopy. The results using RT-PCR showed that CoCl2 increased the AQP4 and VEGF mRNA expression in Muuml;ller cells in a dose and time dependent manner (r=0.952, 0.954;P＜0.05). The expression of AQP4 mRNA in Muuml;ller cells was increased by VEGF (F=12.43,P＜0.05). The expression of AQP4 mRNA was significantly decreased by bevacizumab (F=2 370.37，P＜0.05). Conclusion Bevacizumab can down-regulate the expression of AQP4 mRNA in human Muuml;ller cells under hypoxic conditions partially by VEGF path, which may be a mechanism for treating retinal edema with bevacizumab.
Objective To develop a method for the primary culture of retinal Muuml;ller cells of adult rabbit in vitro. Methods Retina was isolated from adult rabbit, cut into 1 mm times; 1 mm pieces, and placed into Dulbecco modified Eagle medium/F12 containing 20% fetal bovine serum to culture. Cultured cells were identified by inverted phase contrast microscope, transmissim electron microscope and immunohistochemistry staining method. Results Visible cell processes grew out from the retinal tissues after three days culture, and more cells grew radically around the retina after seven days culture. The cultured cells were often inflated at one side and had one long process at another side, and the nuclei were elliptical and there were two or more than two nucleoli under inverted phase contrast microscope. The cytoplasm was rich and contained abundant microfilaments in eight to ten nanometers under transmission electron microscope. Immunohistochemistry assay showed that 95% of the cells were positive for glial fibrillary acidic protein and cellular retinaldehydebinding protein. Conclusion Rabbit retinal Muuml;ller cells can be cultured by the explant culture method.
Objective To investigate the effect of peritoneal exudative cells as feeder cells on growth state of primary culture of adult rat retinal Muuml;ller cells. Methods Peritoneal exudative cells were gained from adult rats, which were identified with specifically biological marker of macrophage (CD68). The phagocytosis was evaluated by the ink particles experiment. Retinal Muuml;ller cells of adult rats were cultured by enzyme digestion method, and identified by GFAP and vimentin immunocytochemically. As the feeder cells, peritoneal exudative cells were cocultured with Muuml;ller cells. The proliferation cycle of Muuml;ller cells was assayed by flow cytometry. One-step TUNEL staining was employed to detect the apoptotic Muuml;ller cells. Results Over ninety-five percent of rat peritoneal exudative cells were macrophage, which have a favourable phagocytic ability for the ink particles. The primary cultured Muuml;ller cells adhered to the wall of flask and grew fast, with large applanate cell bodies. The third-generation cells grew slowly. After cocultured with feeder cells, the Muuml;ller cells showed more rapid growth rate with more cells in S and G2/M phase(S phase, t=4.172, Plt;0.001; G2/M phase, t=3.562, Plt;0.01） and less apoptotic rate (t=3.804, Plt;0.01). The growing cycle was cut down from 25-30 days to 1822 days for the firstgeneration cells, from 10-15 days to 7-10 days for the second-generation cells. Conclusion It is an effective method to use the peritoneal exudative cells as feeder cells cocultured with primary culture of retinal Muuml;ller cells, which can shorten the culture period of Muuml;ller cells in adult rats.
Objective To observe the influence of interleukin-1beta; (IL-1beta;) on the expression of phosphorylated signal transducers and activators of transcription 3 (pSTAT 3) in rat retinal Muuml;ller cells.Methods For in vitro study cultured Muuml;ller cells were treated with IL-1beta; of different concentrations (0, 0.1, 1, 5 and 10 ng/ml) for 24 hours. For in vivo study, 32 Sprague-Dawley（SD）rats were divided into 4 groups randomly (control group，100，500 and 1000 ng/ml group) with 8 rats in each group. After 24 hours of injection with phosphate buffered solution (PBS), or 100,500,1000 ng/ml IL-1beta; into the vitreous cavities of the above rats, retinas were harvested. The expressions of pSTAT3 in cultured Muuml;ller cells or treated retinas were evaluated by indirect immunofluorescence and western blotting.Results After 24 hours of incubation without IL-1beta;, pSTAT3 has little expression in cultured Muuml;ller cells, but was upregulated by 1 ng/ml or higher IL-1beta; in a dosagedependent manner (F=46.64, 43.78;P＜0.01). pSTAT3 was not expressed in adult rat retina, but was upregulated by vitreous injection of 100 ng/ml or higher IL-1beta; in a dosagedependent manner (F=73.53,43.70;P＜0.01).pSTAT3 expressed mainly in inner nuclear layer and ganglion cell layer. Doublelabeling showed that there was no costaining of pSTAT3 and glial fibrillary acidic protein (GFAP) in retina of control group, but there were many costained Muuml;ller cells in retinas treated with IL-1beta;.Conclusions Expression of pSTAT3 in Muuml;ller cells could be activated by IL-1beta; which may represent one pathway link to reactive gliosis.
Objective To observe the regulation effect of transforming growth factor alpha (TGFalpha;) on expression of glutamate transporter（GLAST）and ingestion activity of retinal Muuml;ller cells in mice. Methods To take the retinal tissue of Kunming mouse at postnatal 7～10 day, and then cultured Muuml;ller cells according to literature. The 3～4 generation cultured cells of the same primary cell were divided into two groups at random: ① TGFalpha; group: maintained in different concentrations of TGFalpha; as 50, 75, 125 and 150 ng/ml, 3 holes in each concentration;② Control group: cultured by Eagle culture medium which improved from Dulbeccon and contained 20% fetal calf serum. The influence of different concentrations TGFalpha; on GLAST activity in Muuml;ller cells were observed by L-3H-glutamate uptake detection; the expression of GLAST mRNA in Muuml;ller cells was determined by RT-PCR; the expression of GLAST protein was detected with immunocytochemical staining. Results With the increase of TGFalpha; concentration, both L3H glutamate uptake and GLAST mRNA expression were increased. The L-3H-glutamate accumulation had got to the maximum uptake at concentration of 125 ng/ml, which was 266% of that in control group, meanwhile, the expressions of GLAST mRNA also got to the maximum as 4 times of control group. Immunocytochemical staining indicated that the effect of 125ng/ml TGFalpha; on expression of GLAST protein was higher than that in the control group, the differences between two groups were statistically significant (Plt;0.05). Conclusion TGF-alpha; can increase GLAST activity through up-regulating the expression of GLAST mRNA and protein.
Objective To observe the different effect such as high concentration of glucose and high concentration of insulin on GLUT1 of Rabbit Retinal Muuml;ller Cell in vitro. Methods Rabbit retinal Muuml;ller cells were cultured in vitro with suspended constitution，which were divided as the following groups: common control group，high glucose group，insulin group，high glucose combined insulin group. Laser confocal microscope combined with immunocytochemical and fluorescence staining method to quantitatively analyze the expression condition of GLUT1. Results The expression of GLUT1 has been enhanced obviously by high glucose and high insulin，which locates mainly in the cytoplasm that near to the nucleus. Conclusion Rabbit retinal Muuml;ller cells can express GLUT1，and the expression of GLUT1 can be reinforced by high glucose and high insulin. （Chin J Ocul Fundus Dis，2008，24：265-267）
Objective： To observe the effect of estrogen on the expr ession of pigment epithelium derived factor (PEDF) in cultured retinal Muuml;ller cells under the anoxic condition． Methods：After the anoxic retinal Muuml;ll er cells were tre ated with estrogen (E2) with the concentration of 10-6、10-5 and 10-7 mmol/L, t he level of expression of PEDF mRNA and the protein was detected by reverse tran scriptionpolymerase chain reaction and Western blotting analysis. Results：Th e expression of PEDF mRNA and protein decreased 24 hours after anoxia. E2 with t he concentration of 10-5 and 10-6 mmol/L inhibited the decrease of expression of PEDF mRNA and protein induced by anoxia, which related to the concentration of E2． Conclusion：strogen can regulate the expression of PEDF, which ma y play an important role in the regulation of retinal neovascularization．