本文選題：宮頸癌　切入點：PD-1　出處：《新疆醫(yī)科大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：目的:(1)通過對宮頸鱗癌和宮頸良性疾病組織、配對血液標(biāo)本檢測,了解血液PD-1、組織PD-L1的表達(dá)水平,結(jié)合臨床資料,評價PD-1、PD-L1與宮頸鱗癌患者預(yù)后的關(guān)系;(2)以Wistar鼠Hela細(xì)胞移植瘤為模型,了解放射治療對Wistar鼠PD-1、PD-L1、EBI3的影響;(3)通過對Hela細(xì)胞系過表達(dá)EBI3或抑制EBI3表達(dá),了解與之共培養(yǎng)的T、Treg細(xì)胞增殖、凋亡、細(xì)胞周期的變化,以及PD-1/PD-L1的變化。方法:(1)收集宮頸鱗癌及宮頸良性病變組織標(biāo)本各70例,以及配對的外周血鱗癌標(biāo)本24例,良性病變30例。采用免疫組化法檢測組織PD-L1的表達(dá),流式細(xì)胞術(shù)檢測外周血CD4+PD-1+、CD8+PD-1+亞群細(xì)胞比例。以上資料與患者的臨床病理特征、生存資料做統(tǒng)計分析。(2)對Wistar鼠前肢腋背部接種Hela細(xì)胞,建立皮下移植瘤模型,將建模成功的43只大鼠分為放療組(n=31)和對照組(n=12)。將放療組給予10Gy 6MeV-電子線一次性放療。放療組和對照組分別在放療前、放療后1周和放療后1個月采鼠尾靜脈血和腫瘤組織。外周血Ficoll分離后,在流式細(xì)胞儀上測CD4、CD8計數(shù)。取腫瘤組織用免疫組化法測EBI3、PD-1和PD-L1的表達(dá)。(3)通過慢病毒質(zhì)粒轉(zhuǎn)染的方法建立EBI3過表達(dá)和EBI3抑制性Hela細(xì)胞系,與Wistar大鼠脾臟來源Treg、T細(xì)胞共同培養(yǎng)72小時,使用Western Blot法檢測EBI3過表達(dá)組、EBI3抑制組、陰性對照組、空白轉(zhuǎn)染組細(xì)胞中EBI3、PD-1、PD-L1表達(dá)水平。使用MTT法,以光密度值比較各組Treg細(xì)胞增殖情況。采用流式細(xì)胞術(shù)檢測Treg、CD4+T、CD8+T細(xì)胞的凋亡率,CD4+T和CD8+T細(xì)胞占CD3+T細(xì)胞比率。結(jié)果:(1)兩組人群除腫瘤家族史外,發(fā)病年齡、結(jié)婚年齡、孕產(chǎn)次數(shù)、初潮年齡、是否絕經(jīng)、職業(yè)、文化程度、民族方面都沒有差別。宮頸鱗癌患者外周血PD-1表達(dá)量與組織PD-L1表達(dá)呈正相關(guān)(R2=0.734,P=0.000;R2=0.66,P=0.000)。宮頸良性病變外周血CD4+PD-1+T細(xì)胞、CD8+PD-1+T細(xì)胞與相應(yīng)組織PD-L1的表達(dá)相關(guān)性不大(R2=0.138,P=0.043;R2=0.174,P=0.022)。血CD8+PD-1+T細(xì)胞、組織PD-L1+陽性的細(xì)胞數(shù)在宮頸腫瘤組與宮頸良性病變組具有統(tǒng)計學(xué)差異(p=0.000,p=0.002)。cd8+pd-1表達(dá)與是否絕經(jīng)有統(tǒng)計學(xué)差異(卡方=7.152,p=0.012)。腫瘤大小、分化程度、深肌層侵犯、均與外周血pd-1表達(dá)程度無關(guān)。隨腫瘤分期升高,外周pd-1+的cd4+、cd8+細(xì)胞百分比也相應(yīng)升高。隨腫瘤直徑增大、分期升高,腫瘤組織pd-l1表達(dá)量也相應(yīng)升高。cd4+pd-1+低表達(dá)、組織中pd-l1低表達(dá)的患者生存期較長。多因素分析結(jié)果顯示,組織pd-l1表達(dá)對患者生存有影響(b=1.844,p=0.019)。(2)wistar鼠經(jīng)過放療后,腫瘤直徑較治療前縮小,但是放療后1月部分腫瘤繼續(xù)生長。wistar鼠放療后1月放療組的cd4、cd8細(xì)胞比例較放療前和放療后1周明顯下降,且明顯低于對照組。wistar鼠ebi3、pd-1和pd-l1在放療前、放療后1周無顯著性差異(p0.05)。放療后1月ebi3表達(dá)量有明顯下降,pd-1和pd-l1有明顯增加(p0.05)。對ebi3、pd-1、pd-l1表達(dá)量和t細(xì)胞數(shù)量進(jìn)行相關(guān)性分析提示,ebi3與cd4+、cd8+細(xì)胞數(shù)量呈正相關(guān)(r=0.723,p0.001;r=0.413,p=0.021)。相反,pd-1表達(dá)量與cd4+、cd8+細(xì)胞數(shù)量呈負(fù)相關(guān)(r=-0.631,p0.001;r=-0.509,p=0.004)。pd-l1表達(dá)量也與cd4+、cd8+細(xì)胞數(shù)量呈負(fù)相關(guān)(r=-0.606,p0.001;r=-0.560,p=0.001)。(3)hela細(xì)胞系試驗表明,ebi3過表達(dá)組ebi3表達(dá)升高,pd-1、pd-l1表達(dá)下調(diào)(p0.05),ebi3抑制組ebi3表達(dá)下降,pd-1、pd-l1表達(dá)上升�？瞻邹D(zhuǎn)染組和陰性對照組兩組間的pd-1、pd-l1表達(dá)水平無顯著差異(p0.05)。ebi3過表達(dá)組treg細(xì)胞的od值升高(0.43±0.05),ebi3抑制組treg的od值降低(0.31±0.02)(p0.05)�？瞻邹D(zhuǎn)染組和陰性對照組無差異(p0.05)。ebi3過表達(dá)組、ebi3抑制組、陰性對照組、空白轉(zhuǎn)染組四組在細(xì)胞周期方面沒有明顯差異(p0.05),ebi3過表達(dá)組treg、cd4+t、cd8+t細(xì)胞凋亡率下降(均p0.05),ebi3抑制組treg、cd4+t、cd8+t細(xì)胞凋亡率升高(p均0.05),相同組內(nèi)cd4+t的凋亡低于cd8+t(p均0.05),四組之間cd4/cd8沒有明顯差別(p0.05)。結(jié)論:(1)宮頸鱗癌患者外周血cd4+pd-1+t細(xì)胞、組織pd-l1+陽性的細(xì)胞增多,且與腫瘤的分級、預(yù)后有關(guān),組織pd-l1表達(dá)是患者的獨立預(yù)后因素。(2)放療能使wistar鼠hela細(xì)胞移植腫瘤縮小,提示hela細(xì)胞移植瘤對放療敏感。cd4、cd8細(xì)胞的比例在放療后1月明顯下降,提示t細(xì)胞在放療后受到抑制。放療后1月pd-1和pd-l1表達(dá)增加,提示放療可能造成腫瘤局部微環(huán)境免疫抑制狀態(tài)。放療后1月ebi3表達(dá)下降,提示放療對ebi3表達(dá)有影響。(3)過表達(dá)ebi3,使pd-1、pd-l1表達(dá)下降,treg凋亡下降,t細(xì)胞凋亡下降,反之成立。提示ebi3負(fù)向調(diào)控pd-1、pd-l1表達(dá),負(fù)向調(diào)控treg凋亡及t細(xì)胞凋亡。通過對hela細(xì)胞調(diào)高或降低ebi3表達(dá),發(fā)現(xiàn)treg細(xì)胞周期沒有影響,t細(xì)胞cd4/cd8比例也沒有影響,提示ebi3的功能不包含調(diào)控treg細(xì)胞周期或cd4/cd8細(xì)胞比例。(4)放射治療通過降低宮頸癌細(xì)胞ebi3表達(dá),上調(diào)pd-1/pd-l1通路,下調(diào)t細(xì)胞水平,產(chǎn)生不利的免疫影響;過表達(dá)ebi3,可以抑制PD-1、PD-L1表達(dá),減少T細(xì)胞凋亡,減弱放療引起的免疫抑制狀態(tài)。
[Abstract]:Objective: (1) based on the cervical squamous carcinoma and cervical benign disease tissues, paired blood samples, understand the blood PD-1, the expression level of PD-L1, the evaluation combined with clinical data, PD-1, PD-L1 and prognosis of patients with cervical squamous cell carcinoma; (2) to Wistar rat Hela cells transplanted tumor model, understand PD-L1 on radiation therapy Wistar, EBI3 in PD-1; (3) the Hela cells overexpressing EBI3 or inhibition of EBI3 expression and understanding co cultured with T, Treg cell proliferation, apoptosis, cell cycle changes, and the changes of PD-1/PD-L1. Methods: (1) collection of cervical squamous cell carcinoma and cervical benign lesion tissues in 70 cases, and paired peripheral blood samples of 24 patients with squamous cell carcinoma, 30 cases of benign lesions. The expression of immunohistochemical detection of PD-L1 tissue, peripheral blood CD4+PD-1+ cells were detected by flow cytometry, CD8+PD-1+ cell subsets proportion. In clinical and pathological features of patients with information, students Save the data for statistical analysis. (2) of Wistar mice inoculated with Hela cells forelimb armpit back, a subcutaneous tumor model of 43 rats were divided into model radiotherapy group (n=31) and control group (n=12). The radiotherapy group received 10Gy 6MeV- electronic line one-time radiotherapy. Radiotherapy group and control group before radiotherapy, radiotherapy after 1 weeks and 1 months after radiotherapy by tail vein blood and tumor tissue. The peripheral blood Ficoll after separation, FCM test CD4, CD8 count. The tumor tissue with immunohistochemical staining was used to detect the expression of PD-1 and EBI3, (3) by PD-L1. Methods lentiviral plasmid transfection to establish EBI3 overexpression and EBI3 inhibition of Hela cell line with Wistar rat spleen derived Treg, T cells were co cultured for 72 hours, using the Western Blot method was used to detect the expression of EBI3 group, EBI3 group, negative control group, blank EBI3, sw480i PD-1, the expression level of PD-L1. Use the MTT method to light The density value were compared. The proliferation of Treg cells was detected by Treg, flow cytometry, CD4+T, apoptosis rate of CD8+T cells, CD4+T cells and CD8+T cells accounted for the ratio of CD3+T cells. Results: (1) the two groups in family history of cancer, age of onset, age, number of pregnancy, age of menarche, menopause, occupation there is no difference, culture, nationality. The peripheral blood of patients with cervical carcinoma PD-1 expression and expression of PD-L1 were positively correlated (R2=0.734, P=0.000; R2=0.66, P=0.000). CD4+PD-1+T cells in the peripheral blood of cervical benign lesions, the expression of CD8+PD-1+T cells had little correlation with corresponding tissue PD-L1 (R2=0.138, P=0.043; R2=0.174, P=0.022). Blood CD8+PD-1+T cells, PD-L1+ positive cell number had statistical differences in cervical cancer group and benign cervical lesions group (p=0.000, p=0.002).Cd8+pd-1 expression and whether the vast statistically significant (chi square =7.152, p=0.012 ). The tumor size, degree of differentiation, depth of myometrial invasion, and the degree of expression of PD-1 in peripheral blood. With the tumor stage increased, peripheral pd-1+ cd4+, the percentage of cd8+ cells increased. With the increase of the diameter of the tumor stage, tumor tissue increased, the expression of PD-L1 also increased.Cd4+ pd-1+ low expression, longer period the survival of patients with low expression of PD-L1 in tissues. The results of multivariate analysis showed that the expression of PD-L1 has an effect on the survival of patients (b=1.844, p=0.019). (2) Wistar mice after radiotherapy, tumor diameter reduced after treatment, but after radiotherapy of tumor CD4 in January to January the growth of.Wistar in the radiotherapy group after radiotherapy. The proportion of CD8 cells before and after radiotherapy for 1 weeks was significantly decreased, and significantly lower than the control group ebi3.Wistar rats, PD-1 and PD-L1 before radiotherapy, after radiotherapy 1 weeks had no significant difference (P0.05). The expression of ebi3 after radiotherapy in January has decreased significantly, PD-1 and PD-L1 are Significantly increased (P0.05). The ebi3, PD-1 and T, the amount of cells by correlation analysis suggested that expression of PD-L1, ebi3 and cd4+, the number of cd8+ cells was positively correlated (r=0.723, p0.001; r=0.413, p=0.021). On the contrary, the expression of PD-1 and cd4+, cd8+ was negatively related to the number of cells (r=-0.631, p0.001; r=-0.509, p=0.004) the expression of.Pd-l1 and cd4+, cd8+ was negatively related to the number of cells (r=-0.606, p0.001; r=-0.560, p=0.001). (3) showed that the HeLa cell line test, ebi3 expression increased, ebi3 expression of group PD-1, PD-L1 expression (P0.05), ebi3 inhibition group decreased expression of ebi3, PD-1, PD-L1 expression increased blank. Transfection group and negative control group PD-1 between the two groups, there was no significant difference between the expression level of PD-L1 (P0.05).Ebi3 overexpression group Treg cells increased OD (0.43 + 0.05), ebi3 group Treg inhibited the decrease of OD value (0.31 + 0.02) (P0.05). There is no difference between the blank group and negative control group (transfected the P0.05 expression of.Ebi3 group, EBI) 3 inhibition group, negative control group, blank group four transfection in the cell cycle there is no significant difference (P0.05), ebi3 cd4+t, Treg overexpression group, the apoptosis rate of cd8+t cells decreased (P0.05), ebi3 Treg cd4+t, cd8+t inhibition group, apoptosis rate increased (P 0.05), the same group of apoptosis cd4+t is less than cd8+t (P 0.05), there was no difference in cd4/cd8 between the four groups (P0.05). Conclusion: (1) cd4+pd-1+t cells in the peripheral blood of patients with cervical squamous cell carcinoma tissues, pd-l1+ positive cells increased, which is associated with tumor grade, prognosis, tissue PD-L1 expression is an independent prognostic factor in patients with (2. To reduce Wistar rat) radiotherapy HeLa cell transplantation tumor, suggesting that HeLa is sensitive to radiotherapy.Cd4 cells, CD8 cell ratio in January after radiotherapy was significantly decreased, suggesting that T cells were inhibited after radiotherapy. In January PD-1 and PD-L1 expression increased after radiotherapy, suggesting radiotherapy may cause local tumor microenvironment. Exit immunosuppression in January. Expression of ebi3 decreased after radiotherapy, radiotherapy that influences the expression of ebi3. (3) the over expression of ebi3, PD-1, PD-L1 expression decreased, Treg decreased apoptosis, the apoptosis of T cells decreased, whereas established. Suggesting that ebi3 negatively regulated PD-1, PD-L1 expression, negative regulation of apoptosis and apoptosis of T Treg based on the cells. HeLa cells increased or decreased expression of ebi3, found no effect on the cell cycle of Treg, t also had no effect on the ratio of cd4/cd8 cells, suggesting that ebi3 does not contain the function and regulation of Treg cell cycle or cd4/cd8 cell ratio. (4) radiotherapy of cervical cancer cells by decreasing the expression of ebi3, up regulation of the pd-1/pd-l1 pathway, the down-regulation of T cell level, the adverse impact of immune; overexpression of ebi3 can inhibit the expression of PD-L1, PD-1, T, reduce apoptosis, weaken the immune suppression caused by radiotherapy.

【學(xué)位授予單位】：新疆醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R737.33

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