本文選題：黑素瘤 + 實(shí)時(shí)熒光定量PCR　； 參考：《承德醫(yī)學(xué)院》2012年碩士論文

【摘要】：惡性黑色素瘤是所有惡性腫瘤中發(fā)病率增長(zhǎng)最快的腫瘤之一，近年來受到廣泛重視,其臨床研究也進(jìn)展迅速。黑素瘤的惡性程度高、易復(fù)發(fā)和轉(zhuǎn)移，預(yù)后較差，研究顯示腫瘤厚度3mm的患者5年存活率不足60％，發(fā)生遠(yuǎn)處轉(zhuǎn)移后其平均生存期僅為6—9個(gè)月，5年生存率5％。因此全面深入的研究黑色素瘤發(fā)生、發(fā)展機(jī)制，尋找能夠有效預(yù)測(cè)其生物學(xué)行為的指標(biāo)，對(duì)其早期診斷、指導(dǎo)治療和預(yù)后具有重要的意義。 腎上腺髓質(zhì)素（adrenomedullin,ADM)是內(nèi)源性血管活性肽，是日本學(xué)者于1993年在嗜鉻細(xì)胞瘤組織中發(fā)現(xiàn)的。Miller M J等觀察了肺、腸、乳腺、卵巢、前列腺、腦、軟骨、血液等組織來源的人腫瘤細(xì)胞系，發(fā)現(xiàn)超過90%的腫瘤高表達(dá)ADM，并指出ADM在腫瘤的發(fā)生、發(fā)展中發(fā)揮了重要作用。ADM是否在黑素瘤也高表達(dá)及是否發(fā)揮了一定的生物學(xué)作用，需要研究證實(shí)。 第一部分ADM在惡性黑素瘤中的表達(dá) 目的： 檢測(cè)ADM在人黑素瘤、色素痣組織及黑素瘤細(xì)胞系A(chǔ)375中的表達(dá),以探討ADM在黑素瘤發(fā)生、發(fā)展中的作用。 方法： 用免疫組化法檢測(cè)ADM在人黑素瘤、色素痣組織切片及A375中的表達(dá)情況。 結(jié)果： 惡性黑素瘤組織標(biāo)本中80%檢測(cè)到ADM的表達(dá)，色素痣組織標(biāo)本中僅有43.3%檢測(cè)到ADM的表達(dá)，差異有統(tǒng)計(jì)學(xué)意義（P0.05）。人的成纖維細(xì)胞中ADM為弱陽(yáng)性、A375中ADM呈強(qiáng)陽(yáng)性。 結(jié)論： 腎上腺髓質(zhì)素在人惡性黑素瘤組織及細(xì)胞系A(chǔ)375中高表達(dá)，在人的色素痣和成纖維細(xì)胞中低表達(dá)，提示ADM與惡性黑素瘤發(fā)生、發(fā)展可能密切相關(guān)。 第二部分AMD在黑素瘤中的作用 目的： 探討siRNA沉默腎上腺髓質(zhì)素（adrenomedullin，ADM）基因后對(duì)黑素瘤細(xì)胞系A(chǔ)375增殖、凋亡、周期及侵襲力的影響。 方法： 1.實(shí)驗(yàn)分組:采用人黑素瘤細(xì)胞株A375，利用人工合成的ADM靶向小分子干擾RNA(small interference RAN,siRNA)轉(zhuǎn)染A375細(xì)胞,以抑制ADM的表達(dá)，利用人工合成的非特異序列轉(zhuǎn)染A375細(xì)胞，作為陰性對(duì)照，同時(shí)以正常未轉(zhuǎn)染組細(xì)胞做為空白對(duì)照。 2.通過實(shí)時(shí)熒光定量PCR(qRT-PCR)和免疫細(xì)胞化學(xué)法檢測(cè)處理后各組細(xì)胞ADM的表達(dá)情況,進(jìn)而選擇最有效siRNA。 3.用最有效的siRNA轉(zhuǎn)染A375，分別于24、48h后用MTT法測(cè)各組細(xì)胞的增殖活性。 4.轉(zhuǎn)染48h后，用流式細(xì)胞儀檢測(cè)各組細(xì)胞早期凋亡情況。 5.轉(zhuǎn)染48h后，流式細(xì)胞儀檢測(cè)各組細(xì)胞的周期情況。 6.轉(zhuǎn)染24h后, Transwell測(cè)定沉默ADM對(duì)A375體外侵襲力的影響。 結(jié)果： 1.基因干擾后qRT-PCR篩選有效siRNA結(jié)果：siRNA-1、siRNA-2、siRNA-3干擾率分別為：15%，76%,50%。siRNA-2干擾效果最好。 2.轉(zhuǎn)染后免細(xì)胞化學(xué)法篩選有效siRNA結(jié)果:空白對(duì)照組和轉(zhuǎn)染非特異序列組ADM的染色呈強(qiáng)陽(yáng)性，陽(yáng)性細(xì)胞數(shù)均在90%以上，兩者差異無統(tǒng)計(jì)學(xué)意義(P＞0.05)。siRNA-1、siRNA-2、siRNA-3陽(yáng)性細(xì)胞數(shù)分別為80%,35%,90%。siRAN-2組染色強(qiáng)度明顯弱于其他組。ADMsiRNA-2干擾效果最好。 3.基因干擾后細(xì)胞的增殖率：用ADMsiRAN-2轉(zhuǎn)染A37524、48h后各組OD值示：空白對(duì)照細(xì)胞和陰性對(duì)照細(xì)胞差別無統(tǒng)計(jì)學(xué)意義(P＞0.05)，而轉(zhuǎn)染siRNA的細(xì)胞與前兩者比較差異有統(tǒng)計(jì)學(xué)意義(P＜0.05)。24、48h的抑制率分別為32.2%、31.5%。 4.沉默ADM后A375細(xì)胞的凋亡情況：轉(zhuǎn)染48h后用AnnexinV-EGFP/PI染色在流式細(xì)胞儀檢測(cè)，，結(jié)果顯示：轉(zhuǎn)染siADM的細(xì)胞早期凋亡率（20.20±6.045）%；空白對(duì)照細(xì)胞組的早期凋亡率(1.67±0.340)%；陰性對(duì)照細(xì)胞的早期凋亡率(4.59±1.294)%,轉(zhuǎn)染siADM的細(xì)胞與空白對(duì)照和陰性對(duì)照差異有統(tǒng)計(jì)學(xué)意義(P 0.05),而空白對(duì)照細(xì)胞和陰性對(duì)照細(xì)胞間差異無統(tǒng)計(jì)學(xué)意義(P0.05)。 5.沉默ADM后細(xì)胞DNA周期分布：轉(zhuǎn)染siRNA細(xì)胞的G2/M期細(xì)胞比率為(11.360±1.224)%；明顯高于空白對(duì)照(1.02±0.990)%和陰性對(duì)照組(4.150±1.032)%，差異有統(tǒng)計(jì)學(xué)意義（P0.05），空白對(duì)照與陰性對(duì)照差異無統(tǒng)計(jì)學(xué)意義（P0.05）。而轉(zhuǎn)染siRNA細(xì)胞G1/G0、S期分別是(69.443±2.022)%、(19.197±0.890)%與空白對(duì)照(68.567±3.653)%、(30.417±4.588)%和陰性對(duì)照(70.530±3.008)%、(23.313±4.465)%，差異均無統(tǒng)計(jì)學(xué)意義（P0.05）。 6.沉默ADM基因后A375體外侵襲力檢測(cè)結(jié)果：轉(zhuǎn)染siRNA細(xì)胞的穿膜數(shù)為43.750±3.772，明顯少于空白對(duì)照的70.500±2.723和陰性對(duì)照組的67.500±3.795，差異有統(tǒng)計(jì)學(xué)意義（P0.05）；空白對(duì)照與陰性對(duì)照差異無統(tǒng)計(jì)學(xué)意義（P0.05）。MTT法顯示轉(zhuǎn)染siRNA細(xì)胞的穿膜細(xì)胞的OD值為0.185±0.055，低于空白對(duì)照的0.363±0.006和陰性對(duì)照的0.361±0.086，差異有統(tǒng)計(jì)學(xué)意義（P0.05），空白對(duì)照與陰性對(duì)照差異無統(tǒng)計(jì)學(xué)意義（P0.05）。 結(jié)論: 1沉默ADM基因可抑制A375細(xì)胞的生長(zhǎng) 2沉默ADM基因可促進(jìn)A375細(xì)胞的凋亡 3沉默ADM基因主要對(duì)A375細(xì)胞的G2/M期有影響 4沉默ADM基因可降低A375的體外侵襲能力
[Abstract]:Malignant melanoma is one of the fastest growing tumors in all malignant tumors , and has been paid much attention in recent years . The malignant degree of melanoma is high , the recurrence and metastasis are low , the prognosis is poor , the average survival time after distant metastasis is only 6 - 9 months and the 5 - year survival rate is 5 % .

Adrenomedullin ( ADM ) , an endogenous vascular active peptide , was found in human tumor cell lines , such as lung , intestine , mammary gland , ovary , prostate , brain , cartilage , blood and so on . It was found that more than 90 % of human tumor cell lines in lung , intestine , mammary gland , ovary , prostate , brain , cartilage , blood and so on were observed .

Expression of first part ADM in malignant melanoma

Purpose :

The expression of ADM in human melanoma , pigmented nevi tissue and melanoma cell line A375 was detected to investigate the role of ADM in the pathogenesis and development of melanoma .

Method :

The expression of ADM in human melanoma , pigmented nevi tissue sections and A375 was detected by immunohistochemistry .

Results :

The expression of ADM was detected in 80 % of malignant melanoma tissue samples , and only 43 . 3 % of the specimens from the tissue samples detected the expression of ADM , and the difference was statistically significant ( P0.05 ) . The ADM in human fibroblasts was weak positive , and ADM in A375 was strongly positive .

Conclusion :

The high expression of adrenomedullin in human malignant melanoma tissue and cell line A375 , low expression in human pigmented nevi and fibroblasts suggests that ADM is closely related to the occurrence and development of malignant melanoma .

The role of second part AMD in melanoma

Purpose :

6 . The results of the in vitro invasion ability of A375 transfected siRNA cells were 43.750 鹵 3.772 , 70.500 鹵 2.723 and 67.500 鹵 3.795 , respectively , which were significantly lower than those in the negative control group ( P0.05 ) .

Method :

1 . Experimental group : A375 cells were transfected with human melanoma cell line A375 , and A375 cells were transfected with small interference RAN ( siRNA ) targeting small interfering RAN ( siRNA ) . A375 cells were transfected with artificially synthesized non - specific sequences as negative control , while normal untransfected cells were used as blank control .

2.Through real - time fluorescence quantitative PCR ( qRT - PCR ) and immune cell chemical method , the expression of ADM in each group was detected , and then the most effective siRNA was selected .

3 . A375 was transfected with the most effective siRNA , and the proliferation activity of each group was measured by MTT method after 24 and 48 hours respectively .

4 . After 48h , the apoptosis of each group was detected by flow cytometry .

5 . After 48h , flow cytometry was used to detect the cycle of each group of cells .

6 . After 24 h transfection , Transwell was used to determine the effect of silence ADM on invasion of A375 in vitro .

Results :

1 . siRNA - 1 , siRNA - 2 , siRNA - 3 interference ratios were 15 % , 76 % and 50 % , respectively . siRNA - 1 siRNA - 1 , siRNA - 2 and siRNA - 3 had the best interference effect .

2 . The results of siRNA - 1 , siRNA - 2 , siRNA - 3 positive cells were 80 % , 35 % and 90 % , respectively . The results showed that siRNA - 1 , siRNA - 2 , siRNA - 3 positive cells were 80 % , 35 % and 90 % , respectively .

3 . The proliferation rate of cells after gene interference : The OD values of the cells transfected with the ADMsiRAN - 2 transfected A37524 and 48h showed no statistical significance ( P > 0.05 ) . The difference of the cells transfected with siRNA was statistically significant ( P < 0.05 ) . The inhibition rates of 24 and 48 hours were 32 . 2 % and 31.5 % , respectively .

4 . Apoptosis of A375 cells after transfection : 48h after transfection , AnnexinV - EGFP / PI staining was used to detect the apoptosis of A375 cells . The results showed that the apoptosis rate of cells transfected with siADM was 20.20 鹵 6.45 % .
The early apoptotic rate of blank control group was 1.67 鹵 0.340 % .
The early apoptotic rate of negative control cells ( 4.59 鹵 1.294 ) % , the difference between the cells transfected with siADM and the negative control cells was statistically significant ( P 0.05 ) , but there was no significant difference between the blank control cells and the negative control cells ( P0.05 ) .

5 . Cell DNA cycle distribution after silencing ADM : G2 / M cell ratio of transfected siRNA cells was ( 11.360 鹵 1.224 ) % ;
The G1 / G0 and S phase of transfected siRNA cells were ( 69.443 鹵 2.022 ) % , ( 19.197 鹵 4.588 ) % and negative control ( 70.530 鹵 3.008 ) % , ( 23.313 鹵 4.465 ) % , respectively ( P0.05 ) .

To investigate the effects of siRNA silencing adrenomedullin ( ADM ) gene on proliferation , apoptosis , cycle and invasion of melanoma cell line A375 .
There was no significant difference between the blank control and the negative control ( P0.05 ) . The MTT assay showed that the OD value of the cells transfected with siRNA was 0.185 鹵 0.055 , lower than 0.363 鹵 0.006 in the blank control and 0.361 鹵 0.086 in the negative control . There was no significant difference between the blank control and the negative control ( P0.05 ) .

Conclusion :

1 silencing ADM gene inhibits growth of A375 cells

2 silencing ADM gene promotes apoptosis of A375 cells

3 . The gene silencing ADM mainly affects the G2 / M phase of A375 cells .

4 silencing ADM gene can reduce the invasion ability of A375 in vitro

【學(xué)位授予單位】：承德醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R739.5

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相關(guān)期刊論文 前3條

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2 溫雪萍;李艷;黃紹光;顏志軍;王忠慧;丁梅芬;趙家美;;腎上腺髓質(zhì)素在肺癌患者中的表達(dá)及其臨床意義[J];中國(guó)臨床醫(yī)學(xué);2006年06期

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