| Endometriosis is the common disease and frequently occurring illness in child-bearing period women, which could lead to pelvic pain, dysmenorrhoea and infertility and so on, and effect the health of women seriously. Recently, the morbidity is up to 10%-15%. Although EM ,which has the variation shapes and generally changes, is the benign disease, it has the behavior of infiltrating, metabasis and recurring to become obstinate disease. The cause and mechanism are still unidentify. The researchers gradually realize: the abnormal immune function has important effection to morbidity of EM.,especially in breaking the balance of immune state.It makes EM happening and development. Now there are researches indicating the variation in balancing of Th cytokines balance in most patients of EM, especially in late stage. The changing function of mononuclear cell suppress the cytotoxic action of NK cells and T cells. So it could promote grow of ectopic endometria.The research to the subsets of T lymphocytes and cytokines secreting by them is to get the message of the immune state in EM and to find a new method to search the immunotherapy. In EM, the suppressed function of Th1, the breaking of balancing of Th1/Th2,drifting to Th2 make to heighten the level of Th2. And immune defense function is suppressed, so the endometria cells could'nt be cleaned up, and planting and growth.Our prior researches indicate the level of PRL up in all the body and local in EM, the PRL and PRLR changes in site and ectopic. EM accompany with hyperprolactinemia and infertility are thought more highly. while endometrium is the important site in composition PRL outside appendices suprasphenoidalis, So hyperprolactinemia of EM may be the important factor inducing the happening and development of EM. But there is a problem in the relation about PRL and EM's happening and development, PRL and balancing of Th. Now, the studies in Th cytokine and PRL in EM are still less, especially in regulating the balancing of Th1/Th2 by PRL in EM. One aim of this study is to detect the expressing levels of Th1 and Th2 type cytokines and the different concentration of PRL on regulating peripheral blood mononuclear cells (PBMCs) of EM, in order to find the effection which PRL on Th type cytokinesObjective1.To detect the quantities of T lymphocytic and NK cells in peripheral blood in endometriosis.2.To detect cytotoxicity of NK cells in peripheral blood in EMs.3.To investigate the expression of Th1 and Th2 cytokine in peripheral blood of endometriosis.4.To approach the effection of prolactin to Th's balancing of EMs in vitro. Methods1. The quantities of T lymphocytes and NK cells peripheral blood of endometriosis: Twenty-ejght endometriosis samples and twenty samples were collected from infertilitas feminis proven by laparoscopy or patients with hysteromyoma who were done with hysterectomysecretory. Each one was to collect 3ml blood in ulnar vein before operation, then peripheral blood mononuclear cells were separated by density gradient methods. The quantities of T lymphocytic(CD3+) and NK cells'(CD3-CD56+) in peripheral blood in endometrium samples were detected with flow cytometry.2. Seperation NK cells in peripheral blood by MACS , then detection the cytotoxicity of NK cells by LDH.Twenty-ejght endometriosis samples and twenty samples were collected from infertilitas feminis proven by laparoscopy or patients with hysteromyoma who were done with hysterectomysecretory . Each one was to collect 20ml blood in ulnar vein before operation, NK cells were seperated by MACS, detected the purity coefficient of NK cells by flow cytometry, then detected the cytotoxicity of NK cells by LDH.3. To investigate the variation of Th1 and Th2 cytokine in peripheral blood of endometriosis by ELISA.Twenty-ejght endometriosis samples and twenty samples were collected from infertilitas feminis proven by laparoscopy or patients with hysteromyoma who were done with hysterectomysecretory . Each one was to collect 3ml blood in ulnar vein before operation. The supernatant fluid were obtained and preserved below 80℃, then detecting Th1 (IFN-γ),Th2 (IL-4)by ELISA.4. To investigate the variation of Th1 and Th2 cytokine in NK cells in peripheral blood of endometriosis.Twenty-ejght endometriosis samples and twenty samples were collected from infertilitas feminis proven by laparoscopy or patients with hysteromyoma who were done with hysterectomysecretory .Each one was to collect 5ml blood in ulnar vein before operation. mononuclear cells were separated. To detect the expression of Th1 cells(CD3-CD56+ IFN-γ+)and Th2 cells (CD3-CD56+ IL-4+ cells)by flow cytometry.5.Detection the regulation of recombination human prolactin to Th1 and Th2 ecto-cytokine by ELISA.Peripheral bloods of twenty-four dendometriosis were collected with asepsis ,then PBMC were separated with asepsis by density gradient centrifugation. The cells were cultured in 24 bore plank with 5×105 cells per bore. The cells were cocultured with different density of recombinate human prolactin (rhPRL)and blank for 72 hour and supernatant fluid were obtained and preserved below 80℃. To detect the regulation of recombination human prolactin to Th1(IFN-γ) and Th2(IL-4)cytokines in supernatant fluid by ELISA.6. Detection the regulation of recombination human prolactin to Th1 and Th2 ectocytokine by flow cytometry.Peripheral bloods of fifteen dendometriosis were collected with asepsis ,then PBMC were separated with asepsis. The cells were cultured in 24 bore plank with 5×105 cells per bore. The cells were cocultured with different density of recombinate human prolactin (rhPRL)and blank for 72 hour and cells were obtained. To detect the regulation of recombination human prolactin to Th1(IFN-γ) and Th2(IL-4)cytokines in supernatant fluid by flow cytometry.Result1.The quantities of T lymphocytic and NK cells'in peripheral blood in endometriosis. The ratio of CD3+T cells to total lymphocytes in peripheral blood in EM was (70.41±7.86)%,and the ratio has no significant changes to ratio of control group, (71.81±4.30)%(P >0. 05). The ratio of CD3-CD56+ is (4.93±0.87)%, and the ratio has no significant changes to ratio of control group, (5.97±2.15)%(P >0. 05).2. The cytotoxicity of NK cells in peripheral blood in endometriosis. The purity of NK cells separating by MACS separation is up to 97%.When the effective targets ratio are low, of NK cells in EM is low. The kill ratio is up when the effective targets ratio are up. The kill ratio is 49% when the effective targets ratio is 10﹕1,and up to 54% when 20﹕1. The kill ratio of NK cells to K562 cells is 39%-63%.There is significant difference when the cytotoxicity of test group to control group are 5﹕1 and 10﹕1(P<0.05).The cytotoxicity of NK cells in peripheral blood in endometriosis is lower than it in health adult.3. The exprssion of Th1(IFN-γ) and Th2(IL-4) in peripheral blood in endometriosis.The expression of Th1(IFN-γ) and Th2(IL-4) in peripheral blood in endometriosis was detected by ELISA .The result is :the level of IFN-γin peripheral blood in endometriosis is (109.42±27.54)pg/ml ,and is lower than normal control, (154.27±39.12) pg/ml(P<0.05); the level of IL-4 in peripheral blood in endometriosis is (14.47±1.33) pg/ml ,and is more than normal control, (8.08±0.55) pg/ml (P < 0.05). Th1/Th2(IFN-γ/IL-4) ratio standing for the Th's balancing is (3.65±1.59) in EM and lower than control group(7.11±1.37) (P<0.05) .4. To investigate the variation of CD3-CD56+ IFN-γ+ and CD3-CD56+ IL-4+ cytokine in peripheral blood of endometriosis.The variation of CD3-CD56+ IFN-γ+ and CD3-CD56+ IL-4+ cytokine in peripheral blood of endometriosis was detected by FACS. The result is:the expression of CD3-CD56+ IFN-γ+ in peripheral blood in endometriosis is (6.84±2.75)%,significant lower than control group(16.55±7.00)% (P<0.05). The expression of CD3-CD56+ IL-4+ in peripheral blood in endometriosis is (3.62±1.28)%, significant more than control group (1.29±0.55)%(P<0.05).5. The regulation effection by different concentration rhPRL to peripheral blood monouclear cells TH1 and TH2 in peripheral blood in endometriosis.The expression of the regulation effection by different concentration of rhPRL to peripheral blood monouclear cells Th1 and Th2 in peripheral blood in endometriosis is:lower density of rhPRL(12.5ng/ml)could up-regulate the expression of Th1( IFN-γ) ,and the upgrade tendency decrease in the high density, which is down-regulation. The effect of rhPRL to Th2(IL-4)is the same as above, it means lower concentration of rhPRL(12.5ng/ml)could up-regulate the expression of Th2(IL-4), and the upgrade tendency decrease in the high density. which is down-regulation. Th1/Th2(IFN-γ/IL-4) ratio is up to the max in the effection of the lower density of rhPRL(12.5ng/ml), and the ratio is lower in the effection of the higher concentration of rhPRL, with depressant effect.6. The regulation effection by different concentration of rhPRL to Th1 and Th2 inside the cells in peripheral blood in endometriosis.The expression of the regulation effection by different concentration rhPRL to Th1 and Th2 inside the cells in peripheral blood in endometriosis is: lower concentration of rhPRL(12.5ng/ml)could up-regulate the expression of Th1(IFN-γ) inside the PMBC, and the up-regulation effect is no significant in the high concentration;as the same, the regulation effection of rhPRL to Th2(IL-4)depress the expression of IL-4 in low concentration while the depressant effect decrease significantly in high density. Conclusion:There are no differences about the quantities of total T cells and NK cells in peripheral blood in health adult and endometriosis ,but the cytotoxicity of NK cells is decrease in EM;there is a change in the balancing of Th1 and Th2 expressed by NK cells and to show that Th2 is in the ascendant state. The immunologic function which rhPRL to the balancing of Th1 and Th2 inside and outside the cells in endometriosis were displayed by up-regulation and down- regulation effect.
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