本文選題：乳腺癌 + HMGB1��； 參考：《武漢大學》2015年博士論文

【摘要】：第一部分高遷移族蛋白HMGB1的表達與人乳腺癌細胞放射敏感性的關(guān)系研究目的：研究HMGB1的表達對人乳腺癌細胞放射敏感性的影響。方法：構(gòu)建人乳腺癌MCF-7-sh-HMGB1及陰性對照模型MCF-7-NC穩(wěn)轉(zhuǎn)細胞模型。將shRNA進行合成克隆入pGPU6/GFP/Neo shRNA載體中,將合成之后的載體命名為pGPU6/GFP/Neo-HMGB 1和pGPU6/GFP/Neo-shNC。HMGB1低表達和陰性對照穩(wěn)定轉(zhuǎn)染細胞系采用600ug/ml G418篩選5周,挑選克隆擴增獲得,獲得的穩(wěn)定轉(zhuǎn)染細胞命名為MCF-7-shHMGB1和MCF-7-NC,采用RT-PCR和WB檢測其HMGB1的干擾效率,克隆形成實驗檢測MCF-7-shHMGB1和MCF-7-NC細胞系的放射敏感性。結(jié)果：穩(wěn)定轉(zhuǎn)染細胞系的熒光純度為99%,MCF-7-sh-HMGB1與陰性對照組MCF-7-NC和空白親本組相比,mRNA相對表達量為(0.12975+0.0314)vS.(0.9625±0.0476) vs.(1)。.HMGB干擾后其mRNA表達為親本的12.5%,親本細胞與MCF-7-NC間無明顯差異；蛋白表達方面HMGB1顯著低于MCF-7-NC組,克隆形成顯示HMGB1干擾組的放射敏感性顯著提高,MCF-7-NC和MCF-7-shHMGB1的F2為(0.7756±0.0016) vs. (0.5732±0.0031) (p0.01); DO 為(2.7555±0.0810) vs. (2.4807±0.0331*) (p0.05); Dq為(3.1689±0.1431) vs. (0.7225±0.0210*) (p0.05)。結(jié)論：成功構(gòu)建了人乳腺癌MCF-7-sh-HMGB1及MCF-7-NC穩(wěn)轉(zhuǎn)細胞模型,MCF-7-shHMGB1穩(wěn)定下調(diào)HMGB1的表達,MCF-7-NC無明顯下調(diào)；干擾HMGB1的表達導致MCF-7細胞的放射敏感性顯著增強。第二部分下調(diào)IMGB1的表達增強了人乳腺癌細胞放射敏感性的機制研究目的：探討HMGB1調(diào)控人乳腺癌細胞放射敏感性的分子機制,為乳腺癌的防治提供新的有效靶點,從而為臨床治療提供理論依據(jù)。方法：采用穩(wěn)定干擾細胞模型MCF-7-sh-HMGB1及陰性對照模型MCF-7-NC進行機制研究,相對端粒長度采用realtime-PCR,目的蛋白表達采用Western blot,端粒酶活性采用PCR-ELISA。細胞增殖采用CCK-8檢測。細胞周期與凋亡采用流式細胞術(shù)檢測,DNA損傷灶點的表達采用免疫熒光-共聚焦檢測。活性氧ROS采用活性氧檢測試劑盒(熒光法)檢測。結(jié)果：下調(diào)HMGB1的表達增加了MCF-7細胞的放射敏感性,減少了hTERT和cyclinD1的集聚。MCF-7、MCF-7-shHMGB1和MCF-7-NC的相對端粒長度分別為0.8574±0.0812、0.6763±0.0610和1,MCF-7-shHMGB1組與MCF-7-NC相比,相對端粒長度明顯縮短(***p0.001)；與MCF-7組相比結(jié)果也類似(*p0.05)。MCF-7-NC與MCF-7-shHMGB1二者端粒酶活性分別為1.6155±0.1512和1.169±0.0924,干擾HMGB1后導致MCF-7端粒酶活性明顯下降(*p0.05),而且顯著抑制了細胞增殖。在未照射時,MCF-7-NC和MCF-7-shHMGB1細胞的S期的比例分別為(44.090±5.78)%和(29.080±4.234)%；在6Gy照射后6h,12h,24h進行檢測,MCF-7-NC和MCF-7-shHMGB1細胞的G2/M分別為(29.790±3.291)%、(23.324±4.580)%、(28.592±2.673)%VS(34.293±4.231)%、(31.959±3.265)%、(35.653±5.297)%,兩者差異具有顯著性意義,而其余細胞周期間無明顯差異。MCF-7-NC和MCF-7-shHMGB1的凋亡比例分別為(5.00±0.848)%和(25.500±1.272)%,下調(diào)HMGB1增加了凋亡水平(**p0.01)。在早期階段,HMGB1也參與了活性氧的調(diào)控。HMGB1通過改變端粒結(jié)合蛋白的水平來調(diào)控端粒穩(wěn)態(tài),如TPP1、TRF1、TRF2。HMGB1的下調(diào)同時抑制了ATM和ATR信號通路。結(jié)論：以上結(jié)果顯示下調(diào)HMGB1的表達破壞了人乳腺癌的端粒穩(wěn)態(tài),增強了放射敏感性,抑制了DNA損傷修復(fù),抑制了細胞生長、促進其凋亡,HMGB1可能是人乳腺癌放射治療潛在的新靶點。第三部分人乳腺癌組織中HMGB 1的表達與臨床病理特征相關(guān)性分析目的：比較HMGB1在乳腺浸潤性導管癌患者的腫瘤組織與癌旁組織中的表達差異,并分析HMGB1蛋白表達水平與病理分級、淋巴結(jié)轉(zhuǎn)移、總體預(yù)后等相關(guān)臨床病理特征的關(guān)系,為乳腺癌的治療尋找新的靶標。方法：收集武漢大學中南醫(yī)院病理科存檔的72例乳腺癌患者的組織標本切片,入組標準：我院腫瘤科2001-7至2011-8收治的存有完整蠟塊組織的首診乳腺癌患者；未行術(shù)前新輔助化療,所有患者均行病理診斷確診為浸潤性導管癌；接受標準術(shù)后輔助治療；無其他部位原發(fā)腫瘤；隨訪時間截止為2013-1。利用免疫組化法檢測患者癌組織與癌旁組織中HMGB1的表達差異,并分析HMGB1蛋白的表達與乳腺癌患者的年齡、絕經(jīng)狀態(tài)、腫瘤大小、病理分級、激素受體水平、淋巴結(jié)轉(zhuǎn)移、M分期、總體生存狀態(tài)的相關(guān)性。Kaplan-Meier方法分析HMGB1蛋白的表達對累積生存率的影響。結(jié)果：HMGB1的陽性表達與年齡、絕經(jīng)狀態(tài)、病理分級、雌激素受體狀態(tài)、HER-2受體狀態(tài)、淋巴結(jié)轉(zhuǎn)移分期及M分期無明顯相關(guān)性,與腫瘤的增殖能力、孕激素受體狀態(tài)正相關(guān),與累積生存率負相關(guān),具有統(tǒng)計學意義(P0.05)。結(jié)論：HMGB1的表達與乳腺癌的增殖正相關(guān),其高表達促進腫瘤生長,且與預(yù)后負相關(guān),但需要進一步擴大樣本量及延長部分樣本的隨訪時間。
[Abstract]:Part 1 the relationship between the expression of high mobility group protein HMGB1 and the radiosensitivity of human breast cancer cells: Objective: To study the effect of HMGB1 expression on the radiosensitivity of human breast cancer cells. Methods: to construct human breast cancer MCF-7-sh-HMGB1 and negative control model MCF-7-NC stable cell model. ShRNA was synthesized and cloned into pGPU6/GFP/N In the EO shRNA carrier, the synthesized carrier was named pGPU6/GFP/Neo-HMGB 1 and pGPU6/GFP/Neo-shNC.HMGB1 in the low expression and negative control stable transfection cell line for 5 weeks by 600ug/ml G418, and the cloned amplification was selected. The stable transfected cells were named MCF-7-shHMGB1 and MCF-7-NC, and RT-PCR and WB were used to detect the interference of HMGB1. Results: the radiosensitivity of MCF-7-shHMGB1 and MCF-7-NC cell lines was detected by cloning. Results: the fluorescence purity of the stable transfected cell line was 99%. Compared with the negative control group MCF-7-NC and the blank parent group, the relative expression of mRNA was (0.12975+0.0314) vS. (0.9625 + 0.0476) vs. (1)..HMGB interference, and the mRNA was expressed as a parent. In 12.5%, there was no significant difference between the parent cells and MCF-7-NC; the expression of protein HMGB1 was significantly lower than that of the MCF-7-NC group, and the clonal formation showed that the radiosensitivity of the HMGB1 interference group was significantly increased, the F2 of MCF-7-NC and MCF-7-shHMGB1 was (0.7756 + 0.0016) vs. (0.5732 + 0.0031) (P0.01), DO was (2.7555 + 0.0810) vs. (2.4807 + 0.0331*). Dq was (3.1689 + 0.1431) vs. (0.7225 + 0.0210*) (P0.05). Conclusion: successfully constructed the MCF-7-sh-HMGB1 and MCF-7-NC stable cell model of human breast cancer, MCF-7-shHMGB1 stably downregulated the expression of HMGB1, and there was no obvious downregulation of MCF-7-NC. The expression of interference HMGB1 resulted in a significant increase in the radiosensitivity of MCF-7 cells. Second down regulated the expression of IMGB1. The mechanism of enhancing the radiosensitivity of human breast cancer cells is to explore the molecular mechanism of HMGB1 regulating the radiosensitivity of human breast cancer cells, providing new effective targets for the prevention and control of breast cancer, and providing a theoretical basis for clinical treatment. Methods: a stable interfering cell model MCF-7-sh-HMGB1 and a negative control model MCF-7-N are used. The mechanism of C was studied. The relative telomere length was realtime-PCR, the expression of the target protein was Western blot, the activity of telomerase was detected by CCK-8 in PCR-ELISA. cell proliferation. The cell cycle and apoptosis were detected by flow cytometry. The expression of DNA damage focal point was detected by immunofluorescence confocal detection. Reactive oxygen ROS was tested by active oxygen test. Results: the expression of HMGB1 decreased the radiosensitivity of MCF-7 cells and reduced the concentration of hTERT and cyclinD1, and the relative telomere length of MCF-7-shHMGB1 and MCF-7-NC was 0.8574 + 0.0812,0.6763 + 0.0610 and 1 respectively. Compared with MCF-7-NC, the relative telomere length of MCF-7-shHMGB1 group was significantly shorter (***p0.001). Compared with the MCF-7 group, the telomerase activity of *p0.05.MCF-7-NC and MCF-7-shHMGB1 two were 1.6155 + 0.1512 and 1.169 + 0.0924 respectively. After interfering HMGB1, the telomerase activity of MCF-7 decreased significantly (*p0.05), and the cell proliferation was significantly inhibited. The S phase ratio of MCF-7-NC and MCF-7-shHMGB1 cells was 44.090, respectively (44.090). (+ 5.78)% and (29.080 + 4.234)%, 6h, 12h, 24h were detected after 6Gy irradiation. The G2/M of MCF-7-NC and MCF-7-shHMGB1 cells was (29.790 + 3.291)%, (23.324 + 4.580)%, (28.592 + 2.673)%VS (34.293 +%)%, (31.959 + 3.265)%, (31.959 + 3.265)%, and the difference was significant, but there was no significant difference between the remaining cell cycles. The apoptosis ratio of MCF-7-shHMGB1 was (5 + 0.848)% and (25.500 + 1.272)% respectively. Down regulation of HMGB1 increased the level of apoptosis (**p0.01). At the early stage, HMGB1 also participated in the regulation of reactive oxygen species (.HMGB1) by changing the level of telomere binding protein to regulate telomere homeostasis, such as TPP1, TRF1, TRF2.HMGB1 and inhibition of ATM and ATR signaling. Road. Conclusion: the above results show that the expression of HMGB1 down-regulation destroys the telomere homeostasis of human breast cancer, enhances radiosensitivity, inhibits the repair of DNA damage, inhibits cell growth, and promotes its apoptosis. HMGB1 may be a potential new target for radiation therapy in human breast cancer. The expression of HMGB 1 in third human breast cancer tissues and clinical pathology Objective: To compare the difference in the expression of HMGB1 in the tumor tissues of the patients with invasive ductal carcinoma of the breast, and to analyze the relationship between the expression of HMGB1 protein and pathological classification, lymph node metastasis, the overall prognosis and other related clinicopathological features, to find a new target for the treatment of breast cancer. Methods: collect the Wuhan big 72 cases of breast cancer in the pathology department of the central and southern hospital were examined for tissue specimens. The standard of the group: 2001-7 to 2011-8 of the Department of oncology in our hospital were treated with complete paraffin tissue for the first diagnosis of breast cancer patients; no preoperative neoadjuvant chemotherapy was performed. All patients were diagnosed with a dipped ductal carcinoma by pathological diagnosis; the standard postoperative adjuvant treatment was accepted. There was no primary tumor in other parts; the time of follow-up was 2013-1. using immunohistochemical method to detect the difference in the expression of HMGB1 in the cancer tissues and para cancerous tissues, and to analyze the expression of HMGB1 protein and the age, menopause, tumor size, pathological grade, hormone receptor level, lymph node metastasis, M staging, overall survival state of the patients with breast cancer. The correlation.Kaplan-Meier method was used to analyze the effect of HMGB1 protein expression on the cumulative survival rate. Results: the positive expression of HMGB1 was not associated with age, menopause, pathological grade, estrogen receptor status, HER-2 receptor status, lymph node metastasis staging and M staging, and was positively related to the proliferation of tumor and progesterone receptor status. The negative correlation of cumulative survival rate was statistically significant (P0.05). Conclusion: the expression of HMGB1 is positively related to the proliferation of breast cancer. Its high expression promotes tumor growth and has a negative correlation with the prognosis, but it is necessary to further expand the sample size and prolong the follow-up time of some samples.
【學位授予單位】：武漢大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R737.9

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