本文選題：前列腺癌 + 腺病毒　； 參考：《吉林大學(xué)》2014年博士論文

【摘要】：前列腺癌是最常見的男性腫瘤，屬于高發(fā)性癌癥。許多證據(jù)表明，腫瘤壞死因子相關(guān)的凋亡誘導(dǎo)配體（TRAIL）可誘導(dǎo)癌細(xì)胞的凋亡，因此，TRAIL有希望成為前列腺癌治療的生物制劑。但是，目前調(diào)控TRAIL表達(dá)的載體缺少腫瘤細(xì)胞的特異性，因此對(duì)正常細(xì)胞，特別是肝細(xì)胞也具有細(xì)胞毒性作用。本文為解決這個(gè)問題，將miRNA反應(yīng)元件（MRE）miR-143、 miR-145以及miR-122插入腺病毒載體，成功構(gòu)建Ad-TRAIL-M3腺病毒載體來控制TRAIL的表達(dá)。miR-143、miR-145和miR-122在肝細(xì)胞中特異性表達(dá)，而在前列腺癌中表達(dá)水平降低。Ad-TRAIL-M3將TRAIL以MRE調(diào)控方式表達(dá)，從而在前列腺癌中特異性表達(dá)高水平TRAIL，通過誘導(dǎo)凋亡抑制前列腺癌細(xì)胞的生長(zhǎng)，而在正常細(xì)胞中沒有表達(dá)。隨后在PC-3移植瘤小鼠的研究中，更進(jìn)一步證明了Ad-TRAIL-M3可抑制腫瘤的生長(zhǎng)并且不具有細(xì)胞毒性，具有較高的生物安全性。這個(gè)基于miRNA的基因治療有希望成為前列腺癌治療方法。 具體研究如下： 1.實(shí)驗(yàn)路線 1.1檢測(cè)miR-143、miR-145和miR-122在原發(fā)性前列腺癌和相應(yīng)的非腫瘤前列腺組織、前列腺癌細(xì)胞株、正常前列腺細(xì)胞株和肝細(xì)胞中的表達(dá)量。 1.1.1人正常前列腺上皮細(xì)胞株P(guān)NT2，人正常肝細(xì)胞L-02，激素非依賴性前列腺癌PC-3和激素依賴性前列腺癌LNCaP細(xì)胞培養(yǎng)。 1.1.2通過qPCR比較上述兩種正常細(xì)胞系和人兩種前列腺癌細(xì)胞系中miR-143、miR-145和miR-122表達(dá)水平的差異。 1.1.3收集患者的前列腺癌標(biāo)本、新鮮癌組織及非癌變前列腺組織。1.1.4通過qPCR檢測(cè)比較前列腺癌組織和正常前列腺組織中miR-143、miR-145和miR-122表達(dá)水平的差異。 1.2檢測(cè)miR-143、miR-145和miR-122的MRE是否可以用于在前列腺癌細(xì)胞中表達(dá)目標(biāo)基因 1.2.1將miR-143、miR-145和miR-122的MRE拷貝到熒光素酶的載體編碼區(qū)的下游部位，構(gòu)建psiCheck2-3MRE。1.2.2將psiCheck2和psiCheck2-3MRE轉(zhuǎn)染前列腺癌細(xì)胞，48小時(shí)后用雙熒光素酶報(bào)告基因系統(tǒng)確定其熒光素酶活性。 1.3miR-143、miR-145和miR-122的MRE是否限制前列腺癌細(xì)胞中腺病毒調(diào)控的TRAIL表達(dá) 1.3.1將兩個(gè)miR-143、miR-145和miR-122的MRE拷貝連接進(jìn)腺病毒空載構(gòu)建了含MRE的TRAIL表達(dá)載體，Ad-TRAIL-M3。 1.3.2將Ad-TRAIL和Ad-TRAIL-M3轉(zhuǎn)染PNT2、L-02、PC-3和LNCaP后，通過免疫印跡法檢測(cè)TRAIL的表達(dá)。 1.3.3將Ad-TRAIL和Ad-TRAIL-M3轉(zhuǎn)染PNT2、L-02、PC-3和LNCaP后，通過ELISA檢測(cè)TRAIL的表達(dá)。 1.4檢測(cè)Ad-TRAIL-M3調(diào)控TRAIL表達(dá)是否與miR-143、miR-145和miR-122的表達(dá)量相關(guān) 1.4.1將合成的miRNA抑制劑和模擬物加入到培養(yǎng)的L-02和PC-3細(xì)胞中。通過免疫印跡法檢測(cè)TRAIL的表達(dá)量。 1.5Ad-TRAIL-M3是否能在對(duì)正常細(xì)胞沒顯著毒性的情況下殺死前列腺癌細(xì)胞 1.5.1將Ad-TRAIL和Ad-TRAIL-M3轉(zhuǎn)染PNT2、L-02，MTT試驗(yàn)檢測(cè)Ad-TRAIL-M3是否能在對(duì)正常細(xì)胞沒顯著毒性的情況下殺死前列腺癌細(xì)胞。 1.6Ad-TRAIL-M3是否能選擇性的誘導(dǎo)前列腺癌細(xì)胞凋亡 1.6.1將Ad-TRAIL和Ad-TRAIL-M3轉(zhuǎn)染L-02和PC-3細(xì)胞，用流式細(xì)胞儀檢測(cè)細(xì)胞周期，分析Ad-TRAIL-M3是否能選擇性的誘導(dǎo)前列腺癌細(xì)胞凋亡。 1.6.2免疫印跡法檢測(cè)活化型caspase3和PARP，確定Ad-TRAIL-M3感染細(xì)胞的凋亡激活的信號(hào)通路。 1.7Ad-TRAIL-M3在體內(nèi)對(duì)PC-3前列腺癌是否具有抑制作用 1.7.1通過腫瘤部位注射Ad-TRAIL、Ad-TRAIL-M3、Ad-EGFP和PBS，并定期測(cè)量腫瘤直徑。 1.7.2免疫組化分析腫瘤切片分析TRAIL在PC-3移植瘤中表達(dá)情況。 1.8Ad-TRAIL-M3是否能保護(hù)肝細(xì)胞，免受TRAIL誘導(dǎo)的細(xì)胞毒的影響 1.8.1尾靜脈給予20只正常小鼠注射Ad-TRAIL、Ad-TRAIL-M3、Ad-EGFP和PBS（n=5），隨后收集血樣，檢測(cè)ALT（肝損傷標(biāo)志物）。 2.結(jié)果 2.1miR-143、miR-145和miR-122在前列腺癌細(xì)胞中表達(dá)下調(diào) 實(shí)驗(yàn)結(jié)果顯示，與非腫瘤組織相比，在原發(fā)性前列腺癌樣本中，miR-143、miR-145和miR-122表達(dá)下調(diào)。同樣，與正常前列腺上皮細(xì)胞PNT2和胚胎肝細(xì)胞L-02相比，在前列腺細(xì)胞株P(guān)C-3和LNCaP中，miR-143和miR-145表達(dá)也降低。miR-122在L-02細(xì)胞中高度表達(dá)，在前列腺癌細(xì)胞細(xì)胞株中表達(dá)也降低。 2.2miR-143、miR-145和miR-122的MRE限制外源基因在前列腺癌細(xì)胞中的表達(dá) 實(shí)驗(yàn)結(jié)果顯示，與轉(zhuǎn)染psiCheck2組相比，轉(zhuǎn)染psiCheck2-M3的PNT2中熒光強(qiáng)度被強(qiáng)烈抑制。于此相反，在轉(zhuǎn)染psiCheck2-M3和psiCheck2的前列腺癌細(xì)胞中，熒光表達(dá)無顯著差異。 2.3miR-143、miR-145和miR-122的MRE可限制前列腺癌細(xì)胞中腺病毒調(diào)控的TRAIL表達(dá) 免疫印跡和ELISA結(jié)果顯示所有轉(zhuǎn)染Ad-TRAIL的細(xì)胞都表達(dá)TRAIL蛋白，Ad-TRAIL-M3表達(dá)的TRAIL僅在前列腺癌細(xì)胞中表達(dá)。 2.4Ad-TRAIL-M3調(diào)控TRAIL表達(dá)取決于miR-143、miR-145和miR-122的豐度 免疫印跡結(jié)果顯示，在轉(zhuǎn)染miR-143、miR-145和miR-122抑制劑的L-02細(xì)胞中，TRAIL表達(dá)部分恢復(fù)。而在PC-3細(xì)胞中TRAIL表達(dá)降低，miR-143、miR-145和miR-122可被miR-143、miR-145和miR-122模擬物上調(diào)。qPCR分析顯示，miR-143、miR-145和miR-122的豐度，調(diào)控轉(zhuǎn)染Ad-TRAIL-M3的L-02和PC-3細(xì)胞中TRAIL的表達(dá)水平。 2.5Ad-TRAIL-M3在對(duì)正常細(xì)胞沒顯著細(xì)胞毒性的情況下降低前列腺癌細(xì)胞的存活率 MTT試驗(yàn)顯示PNT2和L-02細(xì)胞被Ad-TRAIL強(qiáng)烈抑制，但Ad-TRAIL-M3對(duì)其沒有影響。而Ad-TRAIL和Ad-TRAIL-M3對(duì)前列腺癌細(xì)胞都有生長(zhǎng)抑制作用。 2.6Ad-TRAIL-M3對(duì)前列腺癌細(xì)胞具有凋亡誘導(dǎo)作用，但對(duì)正常細(xì)胞沒有作用 流式細(xì)胞儀分析顯示，Ad-TRAIL轉(zhuǎn)染后，導(dǎo)致L-02和PC-3細(xì)胞G0/G1亞期數(shù)升高。而Ad-TRAIL-M3處理僅升高前列腺癌細(xì)胞G0/G1亞期數(shù)，，對(duì)正常肝細(xì)胞沒有影響。免疫印跡分析活化型caspase3和PARP表明，這兩個(gè)凋亡相關(guān)的標(biāo)志物在轉(zhuǎn)染Ad-TRAIL-M3或Ad-TRAIL的前列腺癌細(xì)胞中特異性被活化。 2.7Ad-TRAIL-M3對(duì)小鼠PC-3移植瘤的生長(zhǎng)抑制作用 結(jié)果顯示，Ad-TRAIL-M3可抑制PC-3移植瘤生長(zhǎng)達(dá)80%左右，Ad-TRAIL-M3和Ad-TRAIL對(duì)腫瘤生長(zhǎng)的抑制作用沒有顯著的區(qū)別。免疫組化分析腫瘤切片顯示，經(jīng)Ad-TRAIL-M3和Ad-TRAIL治療后，TRAIL在PC-3移植瘤中廣泛表達(dá)。 2.8Ad-TRAIL-3MRE可保護(hù)肝臟不受細(xì)胞毒影響 尾靜脈給予20只正常小鼠注射Ad-TRAIL、Ad-TRAIL-M3、Ad-EGFP和PBS（n=5），隨后收集血樣，檢測(cè)ALT（肝損傷標(biāo)志物）結(jié)果顯示，注射Ad-TRAIL的小鼠ALT水平明顯升高，Ad-TRAIL-M3、Ad-EGFP和PBS組小鼠ALT水平無變化。此外，免疫組化數(shù)據(jù)顯示，Ad-TRAIL-M3治療組小鼠肝臟中無TRAIL表達(dá)。免疫印跡檢測(cè)活化PARP顯示，Ad-TRAIL-M3可誘導(dǎo)腫瘤凋亡，但對(duì)正常肝組織沒有影響。 3.結(jié)論 將miRNA反應(yīng)元件（MRE）miR-143、 miR-145以及miR-122插入腺病毒載體，成功構(gòu)建Ad-TRAIL-M3腺病毒載體來控制TRAIL的表達(dá)，Ad-TRAIL-M3將TRAIL以MRE調(diào)控方式表達(dá)，從而在前列腺癌中特異性表達(dá)高水平TRAIL，通過誘導(dǎo)凋亡抑制前列腺癌細(xì)胞的生長(zhǎng)，隨后在PC-3移植瘤小鼠的研究中，更進(jìn)一步證明了Ad-TRAIL-M3可抑制腫瘤的生長(zhǎng)，而在正常細(xì)胞中TRAIL沒有表達(dá)，不具有細(xì)胞毒性，具有較高的生物安全性。
[Abstract]:In order to solve this problem , it has been proved that TRAIL - M3 can inhibit the growth of prostate cancer cells and has no cytotoxicity and high biological safety . The gene therapy based on miRNA is promising to be a treatment method for prostate cancer .

Specific studies are as follows :

1 . Experimental route

1.1 The expression amount of miR - 143 , miR - 145 and miR - 122 in primary prostate cancer and corresponding non - tumor prostate tissue , prostate cancer cell line , normal prostate cell line and liver cell is detected .

1.1 . 1 Human normal prostate epithelial cell line PNT2 , human normal liver cell line L - 02 , hormone - independent prostate cancer PC - 3 , and hormone - dependent prostate cancer LNAs cell culture .

1.1 . 2 Comparison of miR - 143 , miR - 145 , and miR - 122 expression levels in both normal cell lines and human two prostate cancer cell lines was compared by qPCR .

1.1 . 3 The difference in expression levels of miR - 143 , miR - 145 and miR - 122 in prostate cancer tissue and normal prostate tissue was detected by qPCR by collecting prostate cancer specimens from patients , fresh cancerous tissue , and non - cancerous prostate tissue . 1.4 . 4 .

1.2 Detecting whether MREs of miR - 143 , miR - 145 and miR - 122 can be used to express target genes in prostate cancer cells

1.2 . 1 To construct psiCheck2 - 3MREs . 1.2 . 2 transfected prostate cancer cells with psiCheck2 - 3MREs . 1.2 . 2 , and luciferase activity was determined using a dual luciferase reporter gene system after 48 hours of transfection of psiCheck2 - 3MREs . 1.2 . 2 into the downstream site of the vector coding region of luciferase .

1 . Whether the MREs of miR - 143 , miR - 145 and miR - 122 restrict the expression of TRAIL in prostate cancer cells

1.3 . 1 Two miR - 143 , miR - 145 and miR - 122 were ligated into an adenovirus no - load to construct a TRAIL expression vector containing MREs , Ad - TRAIL - M3 .

1.3 . 2 After transfection of Ad - TRAIL and Ad - TRAIL - M3 into PNT2 , L - 02 , PC - 3 , and LNAs , the expression of TRAIL was detected by Western blotting .

1.3 . 3 After transfection of Ad - TRAIL and Ad - TRAIL - M3 into PNT2 , L - 02 , PC - 3 , and LNAs , the expression of TRAIL was detected by ELISA .

1.4 detecting whether the expression of TRAIL expression in Ad - TRAIL - M3 is related to the expression amount of miR - 143 , miR - 145 and miR - 122

1.4 . 1 The synthesized miRNA inhibitor and mimetic were added to the cultured L - 02 and PC - 3 cells . The expression level of TRAIL was detected by Western blotting .

1.5 Ad - TRAIL - M3 can kill prostate cancer cells without significant toxicity to normal cells

1.5 . 1 Ad - TRAIL and Ad - TRAIL - M3 were transfected into PNT2 , L - 02 , and MTT assay was used to detect whether Ad - TRAIL - M3 could kill prostate cancer cells without significant toxicity to normal cells .

1.6Ad - TRAIL - M3 selectively induces apoptosis of prostate cancer cells

1.6 . 1 Ad - TRAIL and Ad - TRAIL - M3 were transfected into L - 02 and PC - 3 cells . Cell cycle was detected by flow cytometry .

1.6 . 2 The activation type caspase3 and parp were detected by Western blotting , and the signal pathway of apoptosis activation of Ad - TRAIL - M3 infected cells was determined .

Inhibitory Effect of 1.7Ad - TRAIL - M3 on PC - 3 Prostate Cancer in vivo

1.7 . 1 Ad - TRAIL , Ad - TRAIL - M3 , Ad - EGFP and PBS were injected through the tumor site and the tumor diameter was measured periodically .

1.7 . 2 The expression of TRAIL in PC - 3 transplanted tumor was analyzed by immunohistochemistry .

1.8 Ad - TRAIL - M3 protects hepatocytes from TRAIL - induced cytotoxicity

1.8 . 1 Ad - TRAIL , Ad - TRAIL - M3 , Ad - EGFP and PBS ( n = 5 ) were injected into the tail vein in 20 normal mice , followed by the collection of blood samples to detect ALT ( liver injury markers ) .

2 . Results

2.1 Expression of miR - 143 , miR - 145 and miR - 122 in Prostate Cancer Cells

Experimental results showed that miR - 143 , miR - 145 , and miR - 122 expression were down - regulated in a sample of primary prostate cancer compared with non - tumor tissues . Similarly , miR - 143 and miR - 145 expression were also reduced in prostate cancer cell lines PC - 3 and LNAs compared with normal prostate epithelial cells PNT2 and L - 02 . Expression of miR - 122 in L - 02 cells was also reduced .

2.2 Expression of miR - 143 , miR - 145 and miR - 122 in Prostate Cancer Cells

The results showed that the fluorescence intensity was strongly inhibited in the PNT2 transfected psiCheck2 - M3 compared with the transfected psiCheck2 group . In contrast , there was no significant difference in fluorescence expression in the transfected psiCheck2 - M3 and psiCheck2 prostate cancer cells .

2.3miR - 143 , miR - 145 and miR - 122 MREs can limit the expression of TRAIL in prostate cancer cells

Western blot and ELISA results show that all cells transfected with Ad - TRAIL express TRAIL protein and TRAIL - M3 expressed TRAIL is only expressed in prostate cancer cells .

2.4Ad - TRAIL - M3 regulates TRAIL expression depending on the abundance of miR - 143 , miR - 145 and miR - 122

Western blot showed that TRAIL expression was partially restored in the L - 02 cells transfected with miR - 143 , miR - 145 and miR - 122 inhibitors , while miR - 143 , miR - 145 , and miR - 122 were up - regulated by miR - 143 , miR - 145 , and miR - 122 . The qPCR analysis showed that miR - 143 , miR - 145 , and miR - 122 were abundant , and that the expression level of TRAIL in L - 02 and PC - 3 cells transfected with Ad - TRAIL - M3 was regulated .

2.5Ad - TRAIL - M3 reduces the survival rate of prostate cancer cells in the absence of significant cytotoxicity to normal cells

MTT assay showed that PNT2 and L - 02 cells were strongly inhibited by Ad - TRAIL , but Ad - TRAIL - M3 had no effect on prostate cancer cells . Ad - TRAIL and Ad - TRAIL - M3 inhibited the growth of prostate cancer cells .

2.6Ad - TRAIL - M3 has an apoptosis - inducing effect on prostate cancer cells , but has no effect on normal cells .

Flow cytometry analysis showed that after Ad - TRAIL transfection , the G0 / G1 substeps of L - 02 and PC - 3 cells were increased . Ad - TRAIL - M3 treatment only increased the G0 / G1 sub - period of prostate cancer cells and had no effect on normal hepatocytes . Western blot analysis of activated caspase3 and parp showed that these two apoptosis - related markers were specifically activated in prostate cancer cells transfected with Ad - TRAIL - M3 or Ad - TRAIL .

Inhibitory effect of 2 . 7Ad - TRAIL - M3 on growth of mouse PC - 3 transplanted tumor

The results showed that Ad - TRAIL - M3 could inhibit the growth of PC - 3 transplanted tumor by about 80 % , Ad - TRAIL - M3 and Ad - TRAIL had no significant difference in tumor growth . After treatment with Ad - TRAIL - M3 and Ad - TRAIL , TRAIL was widely expressed in PC - 3 transplanted tumor .

2.8 Ad - TRAIL - 3MREs protect the liver from cytotoxic effects

Ad - TRAIL , Ad - TRAIL - M3 , Ad - EGFP and PBS ( n = 5 ) were injected into the tail vein in 20 normal mice . Blood samples were collected . The ALT level of Ad - TRAIL - M3 , Ad - EGFP and PBS group was not changed .

3 . Conclusions

The invention further proves that the Ad - TRAIL - M3 can inhibit the growth of the tumor by inducing the apoptosis to inhibit the growth of the prostate cancer cell , and then further proves that the Ad - TRAIL - M3 can inhibit the growth of the tumor in the study of the PC - 3 transplantation tumor mouse , and further proves that the Ad - TRAIL - M3 can inhibit the growth of the tumor , and the TRAIL is not expressed in the normal cell , and has high biological safety .

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.25

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 尹玉;李明;李昊;江燕;曹立宇;張洪福;徐曉春;;6種microRNAs在前列腺癌組織中的表達(dá)[J];中華男科學(xué)雜志;2010年07期

2 呂驥;任善成;孫穎浩;;前列腺癌的轉(zhuǎn)化醫(yī)學(xué)[J];上海醫(yī)學(xué);2012年05期

本文編號(hào)：1738383