本文選題：雌激素 + 孕激素�。� 參考：《南昌大學(xué)》2017年博士論文

【摘要】：目的用性激素E2、P或E2+P作用于體外培養(yǎng)及荷瘤裸鼠的Hela細胞,檢測細胞增殖、細胞周期及侵襲遷移能力的變化情況;初步評價激素干預(yù)對體外培養(yǎng)及荷瘤裸鼠體內(nèi)HeLa細胞生長的影響,為臨床進行宮頸腺癌激素替代治療提供基礎(chǔ)實驗依據(jù)。方法1.采用免疫組化方法檢測體外培養(yǎng)的人宮頸腺癌HeLa細胞雌、孕激素受體的表達;2.不同濃度的E2、P和E2+P分別與HeLa細胞共培養(yǎng),用CCK8檢測各實驗組培養(yǎng)后24小時、48小時及72小時Hela細胞的OD值,以觀察其增殖情況;3.用Transwell板檢測E2、P和E2+P作用48小時后體外培養(yǎng)Hela細胞的細胞遷移、侵襲能力的變化;4.用流氏細胞儀檢測E2、P和E2+P與Hela細胞共培養(yǎng)48小時后細胞周期及調(diào)亡的變化;5.于裸鼠雙側(cè)后肢背部皮下注射Hela細胞懸混液,24小時后分別予E2、P和E2+P腹腔注射治療,每日一次,建立Hela細胞腫瘤的荷瘤裸鼠,并繼續(xù)用E2,P和E2+P分組治療,觀察腫瘤生長情況及裸鼠存活時間的差異;結(jié)果1.用免疫組化二步法檢測Hela細胞Er、Pr表達,發(fā)現(xiàn)體外培養(yǎng)的Hela細胞Er、Pr表達強陽性;2.結(jié)果顯示,E2、P和E2+P三組的10μmol/L、100μmol/L濃度對Hela細胞生長均有抑制作用,尤其是在培養(yǎng)48小時與72小時后對Hela細胞的抑制作用明顯,與對照組相比差異有統(tǒng)計學(xué)意義(p0.05),且藥物濃度更高的100μmol/L組對Hela細胞的抑制作用更強,而三種藥物的其余濃度組(1μmol/L、0.1μmol/L、0.01μmol/L)對Hela細胞的生長影響不明顯,與對照組相比無統(tǒng)計學(xué)意義(p0.05),因此選擇對細胞抑制作用最強的100μmol/l濃度組進行后續(xù)實驗;實驗的各藥物濃度組均未觀察到其對hela細胞生長的促進作用;3.hela細胞加入激素培養(yǎng)48小時后,經(jīng)流式細胞儀檢測空白對照組、e2、p和e2+p組的凋亡率分別為:5.40±2.38%、20.1±2.20%、20.03±1.91%、24.40±2.40%,各組凋亡率增加,與對照組相比差異有統(tǒng)計學(xué)意義(p0.05),實驗各組均未觀察到細胞凋亡減少情況;細胞周期方面,實驗觀察到e2、p、e2+p組均表現(xiàn)為g0/g1期比例升高,s+g2/m期比例下降,與對照組相比,差異有統(tǒng)計學(xué)意義(p0.05)。4、在我們的實驗藥物濃度下(100μmol/l)培養(yǎng)hela細胞48小時,單獨e2或p對hela細胞遷移、侵襲能力無明顯影響,細胞遷移及侵襲數(shù)與對照組相比差異無統(tǒng)計學(xué)意義(p0.05),但e2+p聯(lián)合使用對hela細胞遷移、侵襲能力減弱,遷移、侵襲細胞數(shù)與對照組相比有差異(p0.05);在我們的實驗藥物濃度下未觀察到對細胞遷移、侵襲的促進作用。5.一周后裸鼠成功建立hela細胞荷瘤裸鼠模型,腫瘤接種24小時即分別開始予e2、p和e2+p治療,空白對照組、e2組、p組及e2+p組成瘤率分別是:87.5%(14/16)、100%(15/15)、81.25%(13/16)、100%(16/16),各組間成瘤率無明顯差異(p0.05)。實驗裸鼠用藥82天,空白對照組、e2組、p組及e2+p組的平均腫瘤體積分別是105.89mm3、450.17mm3、107.96mm3及168.94mm3,其中e2與對照組相比,腫瘤體積更大,有統(tǒng)計學(xué)意義(p0.05),各組裸鼠體重之間差異無統(tǒng)計學(xué)意義(p0.05);腫瘤體積最大者出自e2組,e2組的1號鼠有腹腔轉(zhuǎn)移瘤(體重19.5g)、e2+p組的2號和7號鼠分別有頭部及腹腔轉(zhuǎn)移(二者體重分別為15g及9.9g),并且均在81天死亡,轉(zhuǎn)移率分別為12.5%(1/8)及25%(2/8),其中e2組的轉(zhuǎn)移瘤體積為171.5mm3,e2+p的轉(zhuǎn)移瘤體積分別為1070mm3和600mm3,e2+p轉(zhuǎn)移瘤體積明顯更大。結(jié)論性激素e2、p及e2+p聯(lián)合在一定濃度下對體外培養(yǎng)的hela細胞有抑制作用;適當(dāng)溶度的e2+p可能降低體外培養(yǎng)的hela細胞的侵襲、遷移能力;在荷瘤裸鼠體內(nèi),一定濃度e2促進腫瘤生長,e2+p可能促進腫瘤細胞的早期轉(zhuǎn)移。
[Abstract]:Objective to evaluate the effect of sex hormone E2, P or E2+P on Hela cells in vitro and tumor bearing nude mice, to detect the changes of cell proliferation, cell cycle and invasion and migration, and to evaluate the effect of hormone intervention on the growth of HeLa cells in vitro and in vivo in nude mice. Method 1. immunohistochemistry was used to detect the expression of estrogen and progesterone receptor in human cervical adenocarcinoma HeLa cells in vitro, and 2. different concentrations of E2, P and E2+P were co cultured with HeLa cells respectively. CCK8 was used to detect the OD value of Hela cells in 24 hours, 48 hours and 72 hours after culture in each experiment group, to observe the proliferation of HeLa cells, and 3. to detect E2 with Transwell plate. 48 hours after the action of P and E2+P, the cell migration and invasiveness of Hela cells were cultured in vitro. 4. the cell cycle and the change of apoptosis after 48 hours of co culture of E2, P and E2+P with Hela cells were detected by flow cytometer. 5. in the back of the hind limbs of nude mice, Hela cell suspension was injected subcutaneously, and the treatment of E2, P and E2+P intraperitoneal injection after 24 hours. Once a day, the tumor bearing nude mice of Hela cell tumor were established and the E2, P and E2+P groups were continued to be treated to observe the difference between the tumor growth and the survival time of nude mice. Results 1. the Er and Pr expression of Hela cells were detected by immunohistochemical two step method, and the Hela cells in vitro were found to be Er, Pr expression was strong positive; 2. the results showed that E2, P, and three groups of 10 micron, The concentration of 100 mu mol/L could inhibit the growth of Hela cells, especially the inhibition effect on Hela cells after 48 hours and 72 hours, compared with the control group, the difference was statistically significant (P0.05), and the higher concentration of 100 mu mol/L on the inhibition of Hela cells was stronger, while the remaining concentration group of the three drugs (1 mu mol/L, 0.1 mu). The effect of mol/L, 0.01 mu mol/L) on the growth of Hela cells was not obvious, and there was no significant difference compared with the control group (P0.05). Therefore, a follow-up experiment was carried out in the group of 100 mu mol/l which had the strongest inhibitory effect on the cells. All the concentration groups in the experiment did not observe the promotion effect on the growth of HeLa cells, and 3.hela cells added hormone for 48 hours. After the flow cytometry, the apoptosis rate of E2, P and e2+p groups were 5.40 + 2.38%, 20.1 + 2.20%, 20.03 + 1.91% and 24.40 + 2.40% respectively. The apoptosis rate of each group increased, and the difference was statistically significant (P0.05) compared with the control group (P0.05). The cell cycle aspect, the cell cycle aspect, the experiment observed E2, P, e2+p. The proportion of g0/g1 stage and s+g2/m phase decreased in all groups. Compared with the control group, the difference was statistically significant (P0.05).4. In our experimental drug concentration (100 mu mol/l), HeLa cells were cultured for 48 hours, and E2 or P alone had no significant influence on the migration of HeLa cells, and the number of cell migration and invasion was not statistically significant compared with the control group. Learning significance (P0.05), but e2+p combined use of HeLa cell migration, invasion ability weakened, migration, the number of invasive cells compared with the control group is different (P0.05); in our experimental drug concentration, no observation of cell migration, invasion of.5. a week after the establishment of nude mice to establish a tumor tumor nude mice model, tumor inoculation for 24 hours that is. Do not start with E2, P and e2+p treatment. The tumor rate in blank control group, E2 group, P group and e2+p was 87.5% (14/16), 100% (15/15), 81.25% (13/16), 100% (16/16), and there was no significant difference between each group (P0.05). The average tumor volume of the experimental nude mice was 82 days. 168.94mm3, compared with the control group, the tumor volume was larger than the control group (P0.05), and there was no significant difference between the body weight of the nude mice (P0.05). The largest tumor volume was from the E2 group, and the 1 mice in group E2 had abdominal metastasis (weight 19.5g), and the 2 and 7 mice of e2+p group had head and abdominal metastasis respectively (two of them were 15g respectively. The metastatic rate was 12.5% (1/8) and 25% (2/8) at 81 days. The volume of metastatic tumor in group E2 was 171.5mm3, and the volume of metastatic tumor of e2+p was 1070mm3 and 600mm3, and the volume of e2+p metastatic tumor was significantly larger. Conclusion sex hormone E2, P and e2+p were inhibited under one fixed concentration. The degree of e2+p may reduce the invasion and migration of HeLa cells in vitro, and in the tumor bearing nude mice, a certain concentration of E2 promotes the growth of the tumor, and e2+p may promote the early metastasis of tumor cells.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R737.33

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