本文關(guān)鍵詞：PRL對JurkatE 6-1細(xì)胞增殖分化的影響　出處：《承德醫(yī)學(xué)院》2007年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 催乳素 神經(jīng)內(nèi)分泌-免疫 流式細(xì)胞儀 CD69 RT-PCR

【摘要】： 目的: 催乳素(Prolactin,PRL)是垂體后葉分泌的一種重要的神經(jīng)內(nèi)分泌激素,免疫系統(tǒng)是催乳素作用的一個重要的靶點(diǎn)。已經(jīng)發(fā)現(xiàn)催乳素在調(diào)節(jié)T淋巴細(xì)胞介導(dǎo)的免疫應(yīng)答功能方面起著重要的作用。本文探討催乳素活化淋巴細(xì)胞增殖分化的影響,研究催乳素與免疫細(xì)胞活化有關(guān)的協(xié)同刺激信號之間的關(guān)系,以期建立新的淋巴細(xì)胞激活途徑。 方法: 細(xì)胞培養(yǎng) JurkatE 6-1接種于25ml培養(yǎng)瓶中,用含有體積比為10%小牛血清的RPMI1640培養(yǎng),細(xì)胞調(diào)整到終濃度1×106 cells/ml然后接種到48孔板。分抗CD3單抗和抗CD28單抗組、PRL組和抗CD3、CD28單克隆抗體與PRL共刺激組,設(shè)立陰性空白對照組作比較。在溫度37℃、5% CO2、飽和濕度的培養(yǎng)箱中培養(yǎng)孵化72小時。總RNA提取和RT-PCR 用TRIZOL法提取培養(yǎng)細(xì)胞總RNA,RT-PCR擴(kuò)增ZAP-70、LAT和SLP-76中特定的片斷。將所合成cDNA作為PCR模板,LAT、SLP-76、ZAP-70和PRL的25ul RT-PCR體系中,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’。各反應(yīng)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。按以下條件進(jìn)行RT-PCR反應(yīng)95℃5min,95℃30s,53℃30s,72℃30s,最后7 2℃延長5min。取5μl RT-PCR產(chǎn)物在2%的瓊脂糖凝膠中電泳,與100bp DNA Ladder Marker 5ul直接加入。對照觀察結(jié)果并拍照。流式細(xì)胞術(shù) 取刺激培養(yǎng)細(xì)胞72小時后,收集細(xì)胞加入鼠抗人FITC-labeled CD69單克隆抗體,以檢測細(xì)胞表面CD69表達(dá)情況。調(diào)節(jié)細(xì)胞濃度為1X106個·ml-1,取懸液40ul加人測量管中,用冷PBA(等滲磷酸鹽緩沖液加2%牛血清蛋白,加0.1%疊氮酸鈉)洗滌,避光室溫放置15 min,洗滌去未結(jié)合單抗固定細(xì)胞,上流式細(xì)胞儀檢測。每個樣本計數(shù)10000個細(xì)胞,獲取數(shù)據(jù)并進(jìn)行分析比較平均熒光強(qiáng)度(MFI),輸出結(jié)果并分析。 統(tǒng)計分析: 每組實(shí)驗(yàn)先取平均值,各組間作成對t-test。P 0.05認(rèn)為有意義。 結(jié)果: 1.對PRL作用Jurket細(xì)胞系增殖反應(yīng)的結(jié)果分析:比較各組結(jié)果,抗CD3單抗和抗CD28單抗組與空白對照組相比,吸光度分別為0.422±0.0310和0.403±0.0069,組間t檢驗(yàn)比較P 0.05,有統(tǒng)計學(xué)差別,說明抗CD3單抗和抗CD28單抗可以活化細(xì)胞。而PRL組與空白對照組相比,吸光度分別為0.410±0.0005和0.403±0.0069,組間t檢驗(yàn)比較P 0.05,無統(tǒng)計學(xué)差別,說明抗PRL不可以單獨(dú)活化細(xì)胞,如果用抗CD3單抗和抗CD28單抗和PRL共刺激,吸光度分別為0.462±0.0081和0.403±0.0069,組間t檢驗(yàn)比較P 0.05,有統(tǒng)計學(xué)差別,說明抗CD3單抗和抗CD28單抗和PRL共刺激可以充分活化細(xì)胞,并引起細(xì)胞增殖。 2.分析凝膠電泳灰度LAT、ZAP-70、SLP-76、PRL與β-actin的灰度比可知,抗CD3單抗和抗CD28單抗組與空白對照組相比,LAT、ZAP-70、SLP-76與β-actin的灰度比分別為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,組間t檢驗(yàn)比較P 0.05,有統(tǒng)計學(xué)差別,說明抗CD3單抗和抗CD28單抗可以活化刺激細(xì)胞中LAT、ZAP-70、SLP-76的mRNA的增殖。而PRL組與空白對照組相比,分別為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,組間t檢驗(yàn)比較P 0.05,無統(tǒng)計學(xué)差別,說明PRL不可以單獨(dú)活化刺激細(xì)胞中LAT、ZAP-70、SLP-76的mRNA的增殖。如果用抗CD3單抗和抗CD28單抗和PRL共刺激,分別為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,組間t檢驗(yàn)比較P0.05,有統(tǒng)計學(xué)差別,說明抗CD3單抗和抗CD28單抗和PRL共刺激可以充分活化細(xì)胞,并引起細(xì)胞中LAT、ZAP-70、SLP-76的mRNA的增殖。 3.分析Jurkat E6-1細(xì)胞CD69表達(dá)的平均熒光強(qiáng)度(MFI),抗CD3單抗和抗CD28單抗組與空白對照組相比,CD69表達(dá)的平均熒光強(qiáng)度(MFI)分別為60.61±0.48和3.43±0.05,組間t檢驗(yàn)比較P 0.05,有統(tǒng)計學(xué)差別,說明抗CD3單抗和抗CD28單抗可以活化細(xì)胞,細(xì)胞表達(dá)CD69分子。而PRL組與空白對照組相比,CD69表達(dá)的平均熒光強(qiáng)度(MFI)分別為4.14±0.67和3.43±0.05,組間t檢驗(yàn)比較P 大于0.05,無統(tǒng)計學(xué)差別,說明影響細(xì)胞表達(dá)CD69分子沒有差別。如果用抗CD3單抗和抗CD28單抗和PRL共刺激,CD69表達(dá)的平均熒光強(qiáng)度(MFI)分別為67.09±0.74和3.43±0.05,組間t檢驗(yàn)比較P 0.05,有統(tǒng)計學(xué)差別,說明抗CD3單抗和抗CD28單抗和PRL共刺激可以充分活化細(xì)胞,并引起細(xì)胞增殖且細(xì)胞表達(dá)CD69分子。 結(jié)論: 1.本實(shí)驗(yàn)從不同的層面證實(shí)可以利用抗CD3和抗CD28單克隆抗體激活免疫細(xì)胞JurkatE 6-1。與傳統(tǒng)意義上的細(xì)胞活化途徑有顯著的特異性。 2.用抗CD3和抗CD28單抗和PRL共刺激,能充分活化淋巴細(xì)胞,兩者之間存在協(xié)同效應(yīng)機(jī)制。 3.PRL不能單獨(dú)活化刺激Jurkat E 6-1細(xì)胞增殖。 4.發(fā)現(xiàn)在抗CD3和抗CD28單克隆抗體激活免疫細(xì)胞JurkatE 6-1中存在PRL自分泌現(xiàn)象,其機(jī)制有待進(jìn)一步觀察研究。
[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.

【學(xué)位授予單位】：承德醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R329

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