PRL对JurkatE 6-1细胞增殖分化的影响

发布时间：2018-01-09 00:23

本文关键词：PRL对JurkatE 6-1细胞增殖分化的影响　出处：《承德医学院》2007年硕士论文　论文类型：学位论文

【摘要】： 目的: 催乳素(Prolactin,PRL)是垂体后叶分泌的一种重要的神经内分泌激素,免疫系统是催乳素作用的一个重要的靶点。已经发现催乳素在调节T淋巴细胞介导的免疫应答功能方面起着重要的作用。本文探讨催乳素活化淋巴细胞增殖分化的影响,研究催乳素与免疫细胞活化有关的协同刺激信号之间的关系,以期建立新的淋巴细胞激活途径。方法: 细胞培养 JurkatE 6-1接种于25ml培养瓶中,用含有体积比为10%小牛血清的RPMI1640培养,细胞调整到终浓度1×106 cells/ml然后接种到48孔板。分抗CD3单抗和抗CD28单抗组、PRL组和抗CD3、CD28单克隆抗体与PRL共刺激组,设立阴性空白对照组作比较。在温度37℃、5% CO2、饱和湿度的培养箱中培养孵化72小时。总RNA提取和RT-PCR 用TRIZOL法提取培养细胞总RNA,RT-PCR扩增ZAP-70、LAT和SLP-76中特定的片断。将所合成cDNA作为PCR模板,LAT、SLP-76、ZAP-70和PRL的25ul RT-PCR体系中,引物如下: LAT F-primer: 5’-AGG CTC CTA CGA CAG CAC AT -3’, R-primer: 5’-CAG GTA GCC TGG GTT GTG AT -3’,SLP-76 F-primer: 5’-TGT GCA TCA GAG ACC TTT GC -3’,R-primer:5’-GAC CAG AAA TGT GCC ATC CT -3’,ZAP-70 F-primer: 5’-TGG GGA TGA AGT ACC TGG AG -3’,R-primer: 5’-GCT GGC CAG GCT GTA GTA AC -3’。各反应物量如下10xBV buffer 2.5ul,DMSO 1.5ul,2.5um/ldNTP 2.5ul,β-actin F primer 1.0ul, R primer 1.0ul,Template cDNA 1ul,Water 15ul,Taq 0.5ul。按以下条件进行RT-PCR反应95℃5min,95℃30s,53℃30s,72℃30s,最后7 2℃延长5min。取5μl RT-PCR产物在2%的琼脂糖凝胶中电泳,与100bp DNA Ladder Marker 5ul直接加入。对照观察结果并拍照。流式细胞术取刺激培养细胞72小时后,收集细胞加入鼠抗人FITC-labeled CD69单克隆抗体,以检测细胞表面CD69表达情况。调节细胞浓度为1X106个·ml-1,取悬液40ul加人测量管中,用冷PBA(等渗磷酸盐缓冲液加2%牛血清蛋白,加0.1%叠氮酸钠)洗涤,避光室温放置15 min,洗涤去未结合单抗固定细胞,上流式细胞仪检测。每个样本计数10000个细胞,获取数据并进行分析比较平均荧光强度(MFI),输出结果并分析。统计分析: 每组实验先取平均值,各组间作成对t-test。P 0.05认为有意义。结果: 1.对PRL作用Jurket细胞系增殖反应的结果分析:比较各组结果,抗CD3单抗和抗CD28单抗组与空白对照组相比,吸光度分别为0.422±0.0310和0.403±0.0069,组间t检验比较P 0.05,有统计学差别,说明抗CD3单抗和抗CD28单抗可以活化细胞。而PRL组与空白对照组相比,吸光度分别为0.410±0.0005和0.403±0.0069,组间t检验比较P 0.05,无统计学差别,说明抗PRL不可以单独活化细胞,如果用抗CD3单抗和抗CD28单抗和PRL共刺激,吸光度分别为0.462±0.0081和0.403±0.0069,组间t检验比较P 0.05,有统计学差别,说明抗CD3单抗和抗CD28单抗和PRL共刺激可以充分活化细胞,并引起细胞增殖。 2.分析凝胶电泳灰度LAT、ZAP-70、SLP-76、PRL与β-actin的灰度比可知,抗CD3单抗和抗CD28单抗组与空白对照组相比,LAT、ZAP-70、SLP-76与β-actin的灰度比分别为0.427±0.0045、0.563±0.0087、0.461±0.0106和0.419±0.0062、0.525±0.0021、0.432±0.0028,组间t检验比较P 0.05,有统计学差别,说明抗CD3单抗和抗CD28单抗可以活化刺激细胞中LAT、ZAP-70、SLP-76的mRNA的增殖。而PRL组与空白对照组相比,分别为0.422±0.0037、0.526±0.0019、0.440±0.0006和0.419±0.0062、0.525±0.0021、0.432±0.0028,组间t检验比较P 0.05,无统计学差别,说明PRL不可以单独活化刺激细胞中LAT、ZAP-70、SLP-76的mRNA的增殖。如果用抗CD3单抗和抗CD28单抗和PRL共刺激,分别为0.428±0.0025、0.589±0.0083、0.558±0.0057和0.419±0.0062、0.525±0.0021、0.432±0.0028,组间t检验比较P0.05,有统计学差别,说明抗CD3单抗和抗CD28单抗和PRL共刺激可以充分活化细胞,并引起细胞中LAT、ZAP-70、SLP-76的mRNA的增殖。 3.分析Jurkat E6-1细胞CD69表达的平均荧光强度(MFI),抗CD3单抗和抗CD28单抗组与空白对照组相比,CD69表达的平均荧光强度(MFI)分别为60.61±0.48和3.43±0.05,组间t检验比较P 0.05,有统计学差别,说明抗CD3单抗和抗CD28单抗可以活化细胞,细胞表达CD69分子。而PRL组与空白对照组相比,CD69表达的平均荧光强度(MFI)分别为4.14±0.67和3.43±0.05,组间t检验比较P 大于0.05,无统计学差别,说明影响细胞表达CD69分子没有差别。如果用抗CD3单抗和抗CD28单抗和PRL共刺激,CD69表达的平均荧光强度(MFI)分别为67.09±0.74和3.43±0.05,组间t检验比较P 0.05,有统计学差别,说明抗CD3单抗和抗CD28单抗和PRL共刺激可以充分活化细胞,并引起细胞增殖且细胞表达CD69分子。结论: 1.本实验从不同的层面证实可以利用抗CD3和抗CD28单克隆抗体激活免疫细胞JurkatE 6-1。与传统意义上的细胞活化途径有显著的特异性。 2.用抗CD3和抗CD28单抗和PRL共刺激,能充分活化淋巴细胞,两者之间存在协同效应机制。 3.PRL不能单独活化刺激Jurkat E 6-1细胞增殖。 4.发现在抗CD3和抗CD28单克隆抗体激活免疫细胞JurkatE 6-1中存在PRL自分泌现象,其机制有待进一步观察研究。
[Abstract]:Objective:
Prolactin (Prolactin, PRL) is a kind of important neuroendocrine hormone secreted from the posterior pituitary, the immune system is an important target of prolactin effect. It has been shown that PRL plays an important role in the regulation of immune response mediated by T lymphocytes. This paper discuss the impact of prolactin activated lymphocyte proliferation and differentiation. Study on the relationship between prolactin and immune cell activation of costimulatory signal related, in order to establish new lymphocyte activation pathway.
Method:
cell culture
6-1 JurkatE were seeded in 25ml culture flask, with volume ratio of 10% calf serum RPMI1640 cell culture, adjusted to a final concentration of 1 * 106 cells/ml and then inoculated into 48 well plates. Anti CD3 antibody and anti CD28 monoclonal antibody group, PRL group and anti CD3 monoclonal antibody, CD28 and PRL co stimulation group, set up the negative blank the control group for comparison. At a temperature of 37 DEG C, 5% CO2, saturated humidity incubator in 72 hour incubation culture total RNA extraction and RT-PCR.
Using TRIZOL method to extract the total RNA of the cultured cells, RT-PCR amplification of ZAP-70 fragments, specific LAT and SLP-76. The synthesized cDNA as template of PCR, LAT, SLP-76, ZAP-70 and PRL were 25ul in the RT-PCR system, as follows: LAT F-primer: 5 -AGG CTC CTA CGA CAG 'CAC AT -3' R-primer:, 5 '-CAG GTA GCC TGG GTT GTG AT -3 ", SLP-76 F-primer: -TGT GCA TCA GAG ACC 5' TTT GC -3 ', R-primer:5' -GAC CAG AAA TGT GCC ATC CT -3", ZAP-70 F-primer: -TGG GGA TGA AGT ACC 5 'TGG AG -3', '-GCT GGC CAG GCT R-primer: 5 GTA GTA AC -3 ". The reactants. The amount of 10xBV buffer DMSO 1.5ul as 2.5ul, 2.5um/ldNTP, 2.5ul, F primer 1.0ul R beta -actin, primer 1.0ul, Template cDNA 1ul, Water 15ul, Taq 0.5ul. RT-PCR reaction 5min 95 degrees according to the following conditions, 95 degrees 30s, 53 degrees 30s, 72 degrees 30s, 72 degrees 5min. finally extended take 5 mu L RT-PCR products Electrophoresis in 2% agarose gel and direct addition of 100bp DNA Ladder Marker 5ul. Contrast observation and photograph. Flow cytometry
The stimulation of cultured cells after 72 hours, cells were collected with mouse anti human FITC-labeled monoclonal antibody CD69, to detect cell surface expression of CD69. The regulation of cell concentration is 1X106 / ml-1, 40ul adds the suspension from the measuring tube, with cold isotonic phosphate buffer (PBA + 2% bovine serum albumin, plus 0.1% hydrazoic acid sodium) washing, avoid light at room temperature for 15 min, washing to unbound mAb immobilized cells, flow cytometry detection. Each sample count of 10000 cells to obtain data and compared the mean fluorescence intensity (MFI), and analysis the results.
Statistical analysis:
Take the average value of each experimental group, each group between pairs of t-test.P 0.05 that is meaningful.
Result:
Analysis of the proliferative response of Jurket cell line PRL induced the results of 1. groups were compared: the results, compared with anti CD3 antibody and anti CD28 monoclonal antibody group and blank control group, the absorbance was 0.422 + 0.0310 and 0.403 + 0.0069, t test group P 0.05, the difference was statistically significant, that of anti CD3 antibody and anti CD28 monoclonal antibody can be activated cell. PRL group compared with the control group, the absorbance were 0.410 + 0.0005 and 0.403 + 0.0069, t test group P 0.05, there was no statistically significant difference, show that the anti PRL can not separate cell activation, if using anti CD3 monoclonal antibody and anti CD28 monoclonal antibody and PRL co stimulation, absorbance were 0.462 + 0.0081 and 0.403 + 0.0069, t test group P 0.05, the difference was statistically significant, that of anti CD3 antibody and anti CD28 monoclonal antibody and PRL co stimulation can fully activate cells, and induce cell proliferation.
2. gel electrophoresis analysis of ZAP-70, SLP-76, gray LAT, PRL and beta -actin gray ratio shows that the anti CD3 antibody and anti CD28 monoclonal antibody group compared with the control group, LAT, ZAP-70, SLP-76 and gray beta -actin were 0.427 + 0.0045,0.563 + 0.0087,0.461 + 0.0106 and 0.419 + 0.0062,0.525 + 0.0021,0.432 + 0.0028 group. T test comparison of P 0.05, the difference was statistically significant, show that the anti CD3 monoclonal antibody and anti CD28 monoclonal antibody can stimulate cell activation in LAT, ZAP-70, SLP-76 and PRL. The proliferation of mRNA group compared with the control group, were 0.422 + 0.0037,0.526 + 0.0019,0.440 + 0.0006 and 0.419 + 0.0062,0.525 + 0.0021,0.432 + 0.0028, t group test P 0.05, there was no statistically significant difference, indicating that PRL can not separate activation in cells LAT, ZAP-70, SLP-76 on the proliferation of mRNA. If the use of anti CD3 monoclonal antibody and anti CD28 monoclonal antibody and PRL co stimulation were 0.428 + 0.0025,0.589 + 0.008 3,0.558 + 0.0057 and 0.419 + 0.0062,0.525 + 0.0021,0.432 + 0.0028. There was a statistical difference in t test between groups, indicating that anti CD3 mAb and anti CD28 mAb and PRL co stimulation can activate cells and induce proliferation of LAT, ZAP-70 and SLP-76 in cells.
3. analysis of the mean fluorescence intensity of Jurkat expression in E6-1 CD69 cells (MFI), compared with anti CD3 antibody and anti CD28 monoclonal antibody group and blank control group, the expression of CD69 in the mean fluorescence intensity (MFI) were 60.61 + 0.48 and 3.43 + 0.05, t test group P 0.05, the difference was statistically significant, said that anti CD3 monoclonal antibody and anti CD28 monoclonal antibody can activate cells, cells expressing CD69 molecules. PRL group compared with the control group, the expression of CD69 in the mean fluorescence intensity (MFI) were 4.14 + 0.67 and 3.43 + 0.05, t test group P is greater than 0.05, no significant differences that influence the cellular expression of CD69 has no difference if the use of anti CD3 monoclonal antibody and anti CD28 monoclonal antibody and PRL co stimulation, the expression of CD69 in the mean fluorescence intensity (MFI) were 67.09 + 0.74 and 3.43 + 0.05, t test group P 0.05, the difference was statistically significant, that of anti CD3 antibody and anti CD28 monoclonal antibody and PRL co stimulation can fully activate the fine Cell, cell proliferation and cell expression of CD69 molecules.
Conclusion:
1., the experiment proved that the activation of JurkatE cells from immune cells by anti CD3 and anti CD28 monoclonal antibodies can be significantly different from the traditional sense of cell activation pathway from different levels.
2. the co stimulation of anti CD3 and anti CD28 McAbs and PRL can fully activate lymphocytes, and there is a synergistic mechanism between them.
3.PRL could not activate the proliferation of Jurkat E 6-1 cells alone.
4. it was found that there was a PRL autocrine phenomenon in the immune cell JurkatE 6-1 activated by anti CD3 and anti CD28 monoclonal antibodies, and the mechanism needed to be further studied.

【学位授予单位】：承德医学院
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329

【共引文献】