放射治疗对宫颈癌抑制性免疫因素的影响及其调控机制

发布时间：2018-03-10 06:19

本文选题：宫颈癌　切入点：PD-1　出处：《新疆医科大学》2016年博士论文　论文类型：学位论文

【摘要】：目的:(1)通过对宫颈鳞癌和宫颈良性疾病组织、配对血液标本检测,了解血液PD-1、组织PD-L1的表达水平,结合临床资料,评价PD-1、PD-L1与宫颈鳞癌患者预后的关系;(2)以Wistar鼠Hela细胞移植瘤为模型,了解放射治疗对Wistar鼠PD-1、PD-L1、EBI3的影响;(3)通过对Hela细胞系过表达EBI3或抑制EBI3表达,了解与之共培养的T、Treg细胞增殖、凋亡、细胞周期的变化,以及PD-1/PD-L1的变化。方法:(1)收集宫颈鳞癌及宫颈良性病变组织标本各70例,以及配对的外周血鳞癌标本24例,良性病变30例。采用免疫组化法检测组织PD-L1的表达,流式细胞术检测外周血CD4+PD-1+、CD8+PD-1+亚群细胞比例。以上资料与患者的临床病理特征、生存资料做统计分析。(2)对Wistar鼠前肢腋背部接种Hela细胞,建立皮下移植瘤模型,将建模成功的43只大鼠分为放疗组(n=31)和对照组(n=12)。将放疗组给予10Gy 6MeV-电子线一次性放疗。放疗组和对照组分别在放疗前、放疗后1周和放疗后1个月采鼠尾静脉血和肿瘤组织。外周血Ficoll分离后,在流式细胞仪上测CD4、CD8计数。取肿瘤组织用免疫组化法测EBI3、PD-1和PD-L1的表达。(3)通过慢病毒质粒转染的方法建立EBI3过表达和EBI3抑制性Hela细胞系,与Wistar大鼠脾脏来源Treg、T细胞共同培养72小时,使用Western Blot法检测EBI3过表达组、EBI3抑制组、阴性对照组、空白转染组细胞中EBI3、PD-1、PD-L1表达水平。使用MTT法,以光密度值比较各组Treg细胞增殖情况。采用流式细胞术检测Treg、CD4+T、CD8+T细胞的凋亡率,CD4+T和CD8+T细胞占CD3+T细胞比率。结果:(1)两组人群除肿瘤家族史外,发病年龄、结婚年龄、孕产次数、初潮年龄、是否绝经、职业、文化程度、民族方面都没有差别。宫颈鳞癌患者外周血PD-1表达量与组织PD-L1表达呈正相关(R2=0.734,P=0.000;R2=0.66,P=0.000)。宫颈良性病变外周血CD4+PD-1+T细胞、CD8+PD-1+T细胞与相应组织PD-L1的表达相关性不大(R2=0.138,P=0.043;R2=0.174,P=0.022)。血CD8+PD-1+T细胞、组织PD-L1+阳性的细胞数在宫颈肿瘤组与宫颈良性病变组具有统计学差异(p=0.000,p=0.002)。cd8+pd-1表达与是否绝经有统计学差异(卡方=7.152,p=0.012)。肿瘤大小、分化程度、深肌层侵犯、均与外周血pd-1表达程度无关。随肿瘤分期升高,外周pd-1+的cd4+、cd8+细胞百分比也相应升高。随肿瘤直径增大、分期升高,肿瘤组织pd-l1表达量也相应升高。cd4+pd-1+低表达、组织中pd-l1低表达的患者生存期较长。多因素分析结果显示,组织pd-l1表达对患者生存有影响(b=1.844,p=0.019)。(2)wistar鼠经过放疗后,肿瘤直径较治疗前缩小,但是放疗后1月部分肿瘤继续生长。wistar鼠放疗后1月放疗组的cd4、cd8细胞比例较放疗前和放疗后1周明显下降,且明显低于对照组。wistar鼠ebi3、pd-1和pd-l1在放疗前、放疗后1周无显著性差异(p0.05)。放疗后1月ebi3表达量有明显下降,pd-1和pd-l1有明显增加(p0.05)。对ebi3、pd-1、pd-l1表达量和t细胞数量进行相关性分析提示,ebi3与cd4+、cd8+细胞数量呈正相关(r=0.723,p0.001;r=0.413,p=0.021)。相反,pd-1表达量与cd4+、cd8+细胞数量呈负相关(r=-0.631,p0.001;r=-0.509,p=0.004)。pd-l1表达量也与cd4+、cd8+细胞数量呈负相关(r=-0.606,p0.001;r=-0.560,p=0.001)。(3)hela细胞系试验表明,ebi3过表达组ebi3表达升高,pd-1、pd-l1表达下调(p0.05),ebi3抑制组ebi3表达下降,pd-1、pd-l1表达上升。空白转染组和阴性对照组两组间的pd-1、pd-l1表达水平无显著差异(p0.05)。ebi3过表达组treg细胞的od值升高(0.43±0.05),ebi3抑制组treg的od值降低(0.31±0.02)(p0.05)。空白转染组和阴性对照组无差异(p0.05)。ebi3过表达组、ebi3抑制组、阴性对照组、空白转染组四组在细胞周期方面没有明显差异(p0.05),ebi3过表达组treg、cd4+t、cd8+t细胞凋亡率下降(均p0.05),ebi3抑制组treg、cd4+t、cd8+t细胞凋亡率升高(p均0.05),相同组内cd4+t的凋亡低于cd8+t(p均0.05),四组之间cd4/cd8没有明显差别(p0.05)。结论:(1)宫颈鳞癌患者外周血cd4+pd-1+t细胞、组织pd-l1+阳性的细胞增多,且与肿瘤的分级、预后有关,组织pd-l1表达是患者的独立预后因素。(2)放疗能使wistar鼠hela细胞移植肿瘤缩小,提示hela细胞移植瘤对放疗敏感。cd4、cd8细胞的比例在放疗后1月明显下降,提示t细胞在放疗后受到抑制。放疗后1月pd-1和pd-l1表达增加,提示放疗可能造成肿瘤局部微环境免疫抑制状态。放疗后1月ebi3表达下降,提示放疗对ebi3表达有影响。(3)过表达ebi3,使pd-1、pd-l1表达下降,treg凋亡下降,t细胞凋亡下降,反之成立。提示ebi3负向调控pd-1、pd-l1表达,负向调控treg凋亡及t细胞凋亡。通过对hela细胞调高或降低ebi3表达,发现treg细胞周期没有影响,t细胞cd4/cd8比例也没有影响,提示ebi3的功能不包含调控treg细胞周期或cd4/cd8细胞比例。(4)放射治疗通过降低宫颈癌细胞ebi3表达,上调pd-1/pd-l1通路,下调t细胞水平,产生不利的免疫影响;过表达ebi3,可以抑制PD-1、PD-L1表达,减少T细胞凋亡,减弱放疗引起的免疫抑制状态。
[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.

【学位授予单位】：新疆医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.33

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