肾上腺髓质素在黑素瘤中的表达和作用

发布时间：2018-05-14 05:20

本文选题：黑素瘤 + 实时荧光定量PCR　；参考：《承德医学院》2012年硕士论文

【摘要】：恶性黑色素瘤是所有恶性肿瘤中发病率增长最快的肿瘤之一，近年来受到广泛重视,其临床研究也进展迅速。黑素瘤的恶性程度高、易复发和转移，预后较差，研究显示肿瘤厚度3mm的患者5年存活率不足60％，发生远处转移后其平均生存期仅为6—9个月，5年生存率5％。因此全面深入的研究黑色素瘤发生、发展机制，寻找能够有效预测其生物学行为的指标，对其早期诊断、指导治疗和预后具有重要的意义。肾上腺髓质素（adrenomedullin,ADM)是内源性血管活性肽，是日本学者于1993年在嗜铬细胞瘤组织中发现的。Miller M J等观察了肺、肠、乳腺、卵巢、前列腺、脑、软骨、血液等组织来源的人肿瘤细胞系，发现超过90%的肿瘤高表达ADM，并指出ADM在肿瘤的发生、发展中发挥了重要作用。ADM是否在黑素瘤也高表达及是否发挥了一定的生物学作用，需要研究证实。第一部分ADM在恶性黑素瘤中的表达目的：检测ADM在人黑素瘤、色素痣组织及黑素瘤细胞系A375中的表达,以探讨ADM在黑素瘤发生、发展中的作用。方法：用免疫组化法检测ADM在人黑素瘤、色素痣组织切片及A375中的表达情况。结果：恶性黑素瘤组织标本中80%检测到ADM的表达，色素痣组织标本中仅有43.3%检测到ADM的表达，差异有统计学意义（P0.05）。人的成纤维细胞中ADM为弱阳性、A375中ADM呈强阳性。结论：肾上腺髓质素在人恶性黑素瘤组织及细胞系A375中高表达，在人的色素痣和成纤维细胞中低表达，提示ADM与恶性黑素瘤发生、发展可能密切相关。第二部分AMD在黑素瘤中的作用目的：探讨siRNA沉默肾上腺髓质素（adrenomedullin，ADM）基因后对黑素瘤细胞系A375增殖、凋亡、周期及侵袭力的影响。方法： 1.实验分组:采用人黑素瘤细胞株A375，利用人工合成的ADM靶向小分子干扰RNA(small interference RAN,siRNA)转染A375细胞,以抑制ADM的表达，利用人工合成的非特异序列转染A375细胞，作为阴性对照，同时以正常未转染组细胞做为空白对照。 2.通过实时荧光定量PCR(qRT-PCR)和免疫细胞化学法检测处理后各组细胞ADM的表达情况,进而选择最有效siRNA。 3.用最有效的siRNA转染A375，分别于24、48h后用MTT法测各组细胞的增殖活性。 4.转染48h后，用流式细胞仪检测各组细胞早期凋亡情况。 5.转染48h后，流式细胞仪检测各组细胞的周期情况。 6.转染24h后, Transwell测定沉默ADM对A375体外侵袭力的影响。结果： 1.基因干扰后qRT-PCR筛选有效siRNA结果：siRNA-1、siRNA-2、siRNA-3干扰率分别为：15%，76%,50%。siRNA-2干扰效果最好。 2.转染后免细胞化学法筛选有效siRNA结果:空白对照组和转染非特异序列组ADM的染色呈强阳性，阳性细胞数均在90%以上，两者差异无统计学意义(P＞0.05)。siRNA-1、siRNA-2、siRNA-3阳性细胞数分别为80%,35%,90%。siRAN-2组染色强度明显弱于其他组。ADMsiRNA-2干扰效果最好。 3.基因干扰后细胞的增殖率：用ADMsiRAN-2转染A37524、48h后各组OD值示：空白对照细胞和阴性对照细胞差别无统计学意义(P＞0.05)，而转染siRNA的细胞与前两者比较差异有统计学意义(P＜0.05)。24、48h的抑制率分别为32.2%、31.5%。 4.沉默ADM后A375细胞的凋亡情况：转染48h后用AnnexinV-EGFP/PI染色在流式细胞仪检测，，结果显示：转染siADM的细胞早期凋亡率（20.20±6.045）%；空白对照细胞组的早期凋亡率(1.67±0.340)%；阴性对照细胞的早期凋亡率(4.59±1.294)%,转染siADM的细胞与空白对照和阴性对照差异有统计学意义(P 0.05),而空白对照细胞和阴性对照细胞间差异无统计学意义(P0.05)。 5.沉默ADM后细胞DNA周期分布：转染siRNA细胞的G2/M期细胞比率为(11.360±1.224)%；明显高于空白对照(1.02±0.990)%和阴性对照组(4.150±1.032)%，差异有统计学意义（P0.05），空白对照与阴性对照差异无统计学意义（P0.05）。而转染siRNA细胞G1/G0、S期分别是(69.443±2.022)%、(19.197±0.890)%与空白对照(68.567±3.653)%、(30.417±4.588)%和阴性对照(70.530±3.008)%、(23.313±4.465)%，差异均无统计学意义（P0.05）。 6.沉默ADM基因后A375体外侵袭力检测结果：转染siRNA细胞的穿膜数为43.750±3.772，明显少于空白对照的70.500±2.723和阴性对照组的67.500±3.795，差异有统计学意义（P0.05）；空白对照与阴性对照差异无统计学意义（P0.05）。MTT法显示转染siRNA细胞的穿膜细胞的OD值为0.185±0.055，低于空白对照的0.363±0.006和阴性对照的0.361±0.086，差异有统计学意义（P0.05），空白对照与阴性对照差异无统计学意义（P0.05）。结论: 1沉默ADM基因可抑制A375细胞的生长 2沉默ADM基因可促进A375细胞的凋亡 3沉默ADM基因主要对A375细胞的G2/M期有影响 4沉默ADM基因可降低A375的体外侵袭能力
[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 卤 3.772 , 70.500 卤 2.723 and 67.500 卤 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 卤 6.45 % .
The early apoptotic rate of blank control group was 1.67 卤 0.340 % .
The early apoptotic rate of negative control cells ( 4.59 卤 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 卤 1.224 ) % ;
The G1 / G0 and S phase of transfected siRNA cells were ( 69.443 卤 2.022 ) % , ( 19.197 卤 4.588 ) % and negative control ( 70.530 卤 3.008 ) % , ( 23.313 卤 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 卤 0.055 , lower than 0.363 卤 0.006 in the blank control and 0.361 卤 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

【学位授予单位】：承德医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R739.5

【参考文献】