發(fā)布時間：2018-07-03 21:50

  本文選題：宮頸癌 + 端粒保護蛋白　； 參考：《武漢大學》2014年博士論文

【摘要】：第一部分hPOT1在宮頸癌及癌旁組織中的表達及其臨床意義 目的：研究端粒保護蛋白hPOT1在宮頸癌和癌旁正常宮頸組織中的表達差異,以及hPOT1表達與宮頸癌臨床病理特征之間的關系,探討hPOT1在宮頸癌發(fā)生、發(fā)展中的作用。 方法：應用免疫組織化學方法檢測94例宮頸癌組織和12例癌旁正常宮頸組織中hPOT1蛋白的表達情況。參照文獻方法,采用hPOT1陽性指數(shù)(Positive Index, PI)來進行結果判定。比較hPOT1陽性指數(shù)在宮頸癌組織和宮旁正常組織中的差異。并根據(jù)hPOT1陽性指數(shù)將94例宮頸癌組織分為低表達組(≤30%)和高表達組(30%),分析兩組在年齡分布、病理類型、病理分級、T分期以及有無淋巴結轉移方面的統(tǒng)計學差異。 結果：免疫組化檢測結果顯示,hPOT1蛋白主要表達于宮頸癌和正常宮頸組織的細胞核內(nèi),染色呈棕黃色顆粒。hPOT1蛋白在宮頸癌組織中的陽性指數(shù)為31.52±8.95%,明顯高于其在癌旁組織中的陽性指數(shù)(15.50±4.26%,P0.001)。hPOT1蛋白低表達組和高表達組的陽性指數(shù)分別為23.04±6.36%和40.76±6.49%(P0.001)。兩組的年齡分布、病理類型(宮頸鱗癌與非鱗癌)、病理分級(G1-2/G3)、淋巴結轉移與否(N0/N+)均無顯著性差異(P0.05)。但是,在hPOT1高表達組中T2-3期比例(29/45)明顯高于T1期比例(16/45),在hPOT1低表達組中T1期比例(35/49)顯著高于T2-3期比例(14/49),兩組差異有統(tǒng)計學意義(P0.001),表明T分期越晚,hPOT1表達水平越高。 結論：端粒保護蛋白hPOT1表達升高與宮頸癌的發(fā)生、發(fā)展過程密切相關,hPOT1可能成為抗腫瘤治療的潛在靶點。 第二部分抑制hPOT1表達對宮頸癌細胞放射敏感性的影響 目的：建立穩(wěn)定轉染慢病毒載體重組hPOT1-shRNA質(zhì)粒的宮頸癌C33A細胞模型,以此來研究抑制hPOT1表達對C33A細胞放射敏感性的影響,探討hPOT1成為宮頸癌放射增敏靶點的可能性。 方法：構建靶向抑制hPOT1的重組慢病毒載體pGMLV-SC5-hPOT1-shRN A及相應的陰性對照空質(zhì)粒載體pGMLV-SC5-Neg-shRNA,穩(wěn)定轉染C33A細胞,然后分別通過qRT-PCR法和Western blot方法檢測hPOT1mRNA含量和蛋白表達水平的變化；通過克隆形成實驗,運用GraphPad Prism5.0軟件結合單擊多靶模型公式擬合細胞存活曲線,得到SF2、Do、Dq、N值,計算放射增敏比SERSF2和SERDq。 結果：本實驗成功構建了靶向抑制hPOT1的重組慢病毒載體,并成功轉染C33A細胞,建立了穩(wěn)定轉染的實驗組細胞模型C33A/hPOT1-shRNA和相應的陰性對照組細胞模型C33A/Neg-shRNA。經(jīng)qRT-PCR和Western blot方法驗證,C33A/hPOT1-shRNA細胞的hPOT1mRNA相對含量和蛋白表達水平顯著降低(p0.001)�？寺⌒纬蓪嶒灲Y果顯示,C33A/hPOT1-shRNA和C33A/Neg-shRNA細胞的SF2分別為0.380±0.059和0.563±0.072(P=0.0003),Do分別為1.428和1.658,Dq分別為0.983和2.393。抑制hPOT1后得到SERSF2和SERDq值分別為1.482、2.432。 結論：靶向性抑制hPOT1表達能夠增加宮頸癌C33A細胞對放射線的敏感性,hPOT1有可能成為宮頸癌放射增敏的靶點。 第三部分抑制hPOT1表達對宮頸癌細胞放射增敏作用機制的研究 目的：進一步探討抑制hPOT1表達提高C33A細胞放射敏感性的機制。 方法：靶向抑制C33A細胞hPOT1表達后,通過Southern Blot方法檢測穩(wěn)定轉染細胞傳代至8-10代時的平均端粒長度；TRAP-PCR-ELISA方法檢測端粒酶活性；流式細胞術檢測細胞經(jīng)過X線照射后不同時間點的細胞凋亡水平和細胞周期分布的變化；細胞免疫熒光法結合激光共聚焦顯微鏡檢測輻射后細胞核內(nèi)γ-H2AX灶點變化(反映DNA損傷及修復)。 結果：靶向抑制C33A細胞的hPOT1蛋白表達,對端粒酶活性無明顯影響；但導致其子代細胞的平均端粒延長(8.78±0.726kb比7.20±0.551kb,p=0.0055)：并增加了C33A細胞的自發(fā)性凋亡率(4.07±0.52%比5.16±0.67%,t=2.57,p=0.042)和輻射誘導的凋亡率(43.16+3.11%比34.95%+4.56,t=2.97,p=0.025)；降低了輻射后細胞周期G2/M期阻滯的比例(12h點t=5.995,p0.001；24h點t=9.527,p0.001；48h點t=12.81,p0.001),縮短了G2/M期阻滯的持續(xù)時間。通過激光共聚焦顯微鏡觀察輻射后C33A細胞核內(nèi)的γ-H2AX灶點,我們發(fā)現(xiàn)抑制hPOT1蛋白表達導致5Gy X線照射后0.5h γ-H2AX灶點明顯增多(P0.001),到照射后12h,C33A/hPOT1-shRNA細胞核中殘留的γ-H2AX灶點仍明顯多于陰性對照組細胞C33A/Neg-shRNA,表明靶向抑制hPOT1導致輻射誘導的DSB損傷增多且DSB修復能力下降或者延遲。 結論：靶向抑制hPOT1表達增加C33A細胞放射敏感性的機制與端粒酶活性變化無關,而可能與端粒延長但端粒不穩(wěn)定性增加有關。抑制hPOT1表達減弱和縮短了G2/M期阻滯,可能導致未經(jīng)修復或修復不完全的細胞進入有絲分裂期,進一步增加端粒的不穩(wěn)定性,增加細胞凋亡,從而導致放射增敏。另外,輻射后DSB損傷增多和DSB修復能力下降或修復延遲也可能與hPOT1的放射增敏作用有關�？傊�,抑制hPOT1表達導致C33A細胞放射敏感性增加,hPOT1有可能成為宮頸癌放射增敏的重要靶標之一,本研究為尋找放療增敏的新靶標提供了有利的理論依據(jù)。
[Abstract]:Expression and clinical significance of hPOT1 in cervical cancer and paracancerous tissues
Objective: To investigate the difference in the expression of telomere protective protein hPOT1 in cervical cancer and normal cervix adjacent to cancer, and the relationship between the expression of hPOT1 and the clinicopathological features of cervical cancer, and explore the role of hPOT1 in the development of cervical cancer.
Methods: the expression of hPOT1 protein in 94 cervical cancer tissues and 12 normal cervical tissues was detected by immunohistochemistry. The hPOT1 positive index (Positive Index, PI) was used to determine the results with reference to the literature method. The difference between the hPOT1 positive index in the cervical cancer tissue and the normal tissue adjacent to the uterus was compared. And according to hPOT, the difference between the positive index of the positive index of the cervical cancer and the normal tissues near the uterus was compared. In the 1 positive index, 94 cases of cervical cancer were divided into low expression group (less than 30%) and high expression group (30%). The statistical differences in age distribution, pathological type, pathological grade, T staging and lymph node metastasis were analyzed in the two groups.
Results: the results of immunohistochemical staining showed that hPOT1 protein was mainly expressed in the nucleus of cervical cancer and normal cervical tissue. The positive index of brown yellow granules.HPOT1 protein in cervical cancer tissues was 31.52 + 8.95%, which was significantly higher than that of the positive index (15.50 + 4.26%, P0.001) of.HPOT1 protein in the para cancerous tissue. The positive index of the expression group was 23.04 + 6.36% and 40.76 + 6.49% (P0.001). The age distribution of the two groups, pathological type (cervical squamous cell carcinoma and non squamous cell carcinoma), pathological classification (G1-2/G3), lymph node metastasis (N0/N+) had no significant difference (P0.05). However, the proportion of T2-3 phase (29/45) in the high expression group of hPOT1 was significantly higher than that of T1 phase (16/45), in hPOT1. The T1 phase ratio (35/49) in the low expression group was significantly higher than that in the T2-3 phase (14/49), and the difference between the two groups was statistically significant (P0.001), indicating that the later the T stage, the higher the hPOT1 expression level.
Conclusion: the increased expression of telomere protection protein hPOT1 is closely related to the occurrence and development of cervical cancer. HPOT1 may be a potential target for anti-cancer therapy.
Second, inhibition of hPOT1 expression on radiosensitivity of cervical cancer cells.
Objective: to establish a C33A cell model to stabilize the hPOT1-shRNA plasmid transfected with lentivirus carrying body weight group, in order to study the effect of inhibition of hPOT1 expression on the radiosensitivity of C33A cells and to explore the possibility of hPOT1 as a radiosensitizing target of cervical cancer.
Methods: the recombinant lentivirus vector pGMLV-SC5-hPOT1-shRN A and the corresponding negative control empty plasmid vector pGMLV-SC5-Neg-shRNA were constructed, and the C33A cells were transfected stably. Then the changes of hPOT1mRNA content and protein expression level were detected by qRT-PCR and Western blot methods, and the experiment was carried out by cloning and using Graph. Pad Prism5.0 software combined with click multiple target model formula to fit cell survival curve, get SF2, Do, Dq, N value, and calculate radiosensitization ratio SERSF2 and SERDq..
Results: the recombinant lentivirus vector targeting hPOT1 was successfully constructed, and C33A cells were successfully transfected. The cell model C33A/hPOT1-shRNA of the stable transfected experimental group and the corresponding negative control group cell model C33A/Neg-shRNA. were verified by qRT-PCR and Western blot methods, and the hPOT1mRNA of C33A/hPOT1-shRNA cells was relatively contained. The expression level of the quantity and protein was significantly decreased (p0.001). The results of the clone formation experiment showed that the SF2 of C33A/hPOT1-shRNA and C33A/Neg-shRNA cells were 0.380 + 0.059 and 0.563 + 0.072 (P=0.0003), Do was 1.428 and 1.658 respectively. Dq was 0.983 and 2.393. inhibited hPOT1, respectively, and SERSF2 and SERDq were 1.482,2.432..
Conclusion: targeted inhibition of hPOT1 expression can increase the sensitivity of cervical cancer C33A cells to radiation, and hPOT1 may become a target for radiosensitization of cervical cancer.
The third part is to inhibit the radiosensitization mechanism of hPOT1 expression on cervical cancer cells.
Objective: to further explore the mechanism of inhibiting the expression of hPOT1 and enhancing the radiosensitivity of C33A cells.
Methods: after the target inhibition of C33A cell hPOT1 expression, the average telomere length of the stable transfected cells to 8-10 generations was detected by Southern Blot method. The activity of telomerase was detected by TRAP-PCR-ELISA, and the changes of cell apoptosis and cell cycle distribution at different time points after X-ray irradiation were detected by flow cytometry. Cell immunofluorescence combined with confocal laser scanning microscopy was used to detect the changes of the gamma -H2AX foci in the nucleus after irradiation (reflecting DNA damage and repair).
Results: the expression of hPOT1 protein in C33A cells had no significant effect on telomerase activity, but the average telomere prolonged (8.78 + 0.726kb ratio 7.20 + 0.551kb, p=0.0055) in the progeny cells, and increased the spontaneous apoptosis rate of C33A cells (4.07 + 0.52% to 5.16 + 0.67%, t=2.57, p=0.042) and radiation induced apoptosis rate (43.16+3.11) Compared with 34.95%+4.56, t=2.97, p=0.025), the proportion of G2/M block in cell cycle after radiation was reduced (12h point t=5.995, p0.001; 24h point t=9.527, p0.001; 48h t=12.81, etc.), which shortened the duration of the arrest period. White expression led to a significant increase in 0.5h gamma -H2AX focal points (P0.001) after 5Gy X-ray irradiation. After irradiation, the residual gamma -H2AX focal points in the nucleus of 12h and C33A/hPOT1-shRNA were still more than C33A/Neg-shRNA in the negative control group, indicating that the target inhibition hPOT1 led to the increase of the DSB damage induced by the radiation and the decline or delay of the DSB repair ability.
Conclusion: the mechanism of targeting inhibition of hPOT1 expression to increase the radiosensitivity of C33A cells is not related to the changes of telomerase activity, but may be related to telomere extension but increased telomere instability. Inhibition of hPOT1 expression is reduced and G2/M block is shortened, which may lead to unrepaired or repair of incomplete cells into mitosis, and further increase The instability of telomere increases cell apoptosis and causes radiation sensitization. In addition, the increase of DSB damage after radiation and the decrease of DSB repair ability or repair delay may also be related to the radiosensitization of hPOT1. In conclusion, inhibition of hPOT1 expression leads to increased radiosensitivity of C33A cells, and hPOT1 may be an important target for radiosensitization of cervical cancer. This study provides a theoretical basis for finding new targets for radiosensitization.
【學位授予單位】：武漢大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R737.33

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相關期刊論文 前1條

1 王忠民;姜繼勇;喬新民;;復發(fā)宮頸癌發(fā)病因素及其不同治療方式對預后的影響[J];中國實用婦科與產(chǎn)科雜志;2010年03期

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本文編號：2095120