發(fā)布時(shí)間：2018-06-16 00:34

  本文選題：Rab23 + 鱗癌��； 參考：《第四軍醫(yī)大學(xué)》2012年碩士論文

【摘要】：皮膚鱗狀細(xì)胞癌（cutaneous squamous cell carcinoma, cSCC）是皮膚的第二常見腫瘤，發(fā)生率僅次于基底細(xì)胞癌（basal cell carcinoma, BCC）。它是一種侵襲性癌，可早期轉(zhuǎn)移。因此，關(guān)于鱗狀細(xì)胞癌發(fā)生、發(fā)展及其侵襲、轉(zhuǎn)移的研究一直是皮膚科學(xué)界關(guān)注的重點(diǎn)領(lǐng)域之一。 Rab23是RAS原癌基因成員，其編碼的蛋白是小GTP酶超家族Rab家族成員，其突變可引起小鼠開腦綜合征（open brain syndrome）。它參與囊泡轉(zhuǎn)運(yùn)，是sonic hedgehog信號通路的負(fù)調(diào)控因子，并在不同腫瘤中發(fā)揮不同作用。課題組曾發(fā)現(xiàn)Rab23在皮膚鱗癌中高表達(dá)，但目前尚未見Rab23在皮膚鱗癌侵襲中的作用及機(jī)制的研究。本研究通過免疫組化檢測Rab23在光線性角化病、鮑溫病、皮膚鱗癌和正常皮膚中的表達(dá)，分析其與鱗癌侵襲的相關(guān)性，，檢測Rab23在鱗癌細(xì)胞系中的表達(dá)，研究Rab23對鱗癌細(xì)胞侵襲的影響，并初步探討機(jī)制。 實(shí)驗(yàn)方法： ①免疫組化檢測Rab23在光線性角化病、鮑溫病、皮膚鱗狀細(xì)胞癌及正常皮膚中的表達(dá)，統(tǒng)計(jì)分析Rab23在各組中的表達(dá)差異，及Rab23陽性表達(dá)與皮膚鱗癌臨床病理特征的相關(guān)性；②Western blotting檢測Rab23在HSC-2、HSQ-89、Sa-3、Tca四個(gè)鱗癌細(xì)胞系及Hacat中的表達(dá)；③Transwell小室檢測Rab23對鱗癌細(xì)胞侵襲能力的影響；④細(xì)胞劃痕試驗(yàn)檢測Rab23對鱗癌細(xì)胞遷移能力的影響；⑤Real time RT-PCR檢測干涉或過表達(dá)Rab23后鱗癌細(xì)胞中侵襲相關(guān)分子MMP2、MMP9，hedgehog信號通路中Ptch1、Gli1、Gli2的mRNA水平；⑥Western blotting檢測侵襲相關(guān)分子Rac1的表達(dá)。 實(shí)驗(yàn)結(jié)果： ①Rab23在皮膚鱗狀細(xì)胞癌、光線性角化病、鮑溫病及正常皮膚中的表達(dá)：Rab23在光線性角化病、鮑溫病及皮膚鱗狀細(xì)胞癌組織中均有表達(dá)，在正常皮膚中不表達(dá)，非曝光部位及中、低分化鱗癌是Rab23陽性表達(dá)的危險(xiǎn)因素； ②Rab23在HSC-2、HSQ-89、Sa-3、Tca四個(gè)鱗癌細(xì)胞系及Hacat中的表達(dá)：Rab23在四個(gè)鱗癌細(xì)胞系中均有表達(dá)，在HSC-2、HSQ-89細(xì)胞中高表達(dá)，在Sa-3及Tca中低表達(dá)，在Hacat中不表達(dá)； ③Transwell小室檢測Rab23對鱗癌細(xì)胞侵襲能力的影響：HSQ-89細(xì)胞行Rab23特異的siRNA干涉后，細(xì)胞侵襲能力減弱；Sa-3細(xì)胞行質(zhì)粒轉(zhuǎn)染過表達(dá)Rab23后，細(xì)胞侵襲能力較對照組增強(qiáng)； ④細(xì)胞劃痕試驗(yàn)檢測Rab23對鱗癌細(xì)胞遷移能力的影響：HSQ-89細(xì)胞行siRNA干涉后，干涉組與對照組細(xì)胞均僅遷移1個(gè)細(xì)胞直徑距離；Sa-3細(xì)胞行質(zhì)粒轉(zhuǎn)染后，過表達(dá)組與對照組細(xì)胞遷移能力差異無統(tǒng)計(jì)學(xué)意義； ⑤Real time RT-PCR檢測干涉或過表達(dá)Rab23后相關(guān)分子的水平：HSQ-89細(xì)胞Rab23干涉組MMP2、MMP9、Gli2的mRNA水平較對照組低，Ptch1、Gli1的mRNA水平較對照組高；Sa-3細(xì)胞Rab23過表達(dá)組中MMP2、MMP9、Gli2的mRNA水平較對照組高，Ptch1、Gli1較對照組低； ⑥Western blotting檢測干涉或過表達(dá)Rab23后Rac1的表達(dá)：HSQ-89細(xì)胞干涉Rab23表達(dá)后，Rac1水平較對照組低；Sa-3細(xì)胞過表達(dá)Rab23表達(dá)后，Rac1水平較對照組高。 主要結(jié)論： ①Rab23在光線性角化病、鮑溫病及皮膚鱗癌中陽性表達(dá)，而正常皮膚中不表達(dá)，Rab23在鱗癌細(xì)胞系中表達(dá)，Hacat中不表達(dá)，說明其參與皮膚鱗癌的發(fā)生發(fā)展； ②Rab23促進(jìn)鱗癌細(xì)胞的侵襲，不影響細(xì)胞遷移； ③Rab23促進(jìn)鱗癌侵襲可能通過Rac1、MMP2、MMP9，可能不通過hedgehog信號通路。
[Abstract]:Cutaneous squamous cell carcinoma (cSCC) is the second common tumor of the skin, which is only inferior to basal cell carcinoma (basal cell carcinoma, BCC). It is an invasive carcinoma and can be transferred early. Therefore, the study of squamous cell carcinogenesis, development and invasion, and metastasis have always been the concern of the skin science community. One of the key areas.
Rab23 is a member of the RAS proto oncogene and its encoding protein is a member of the small GTP enzyme superfamily Rab family. Its mutation can cause open brain syndrome (open brain syndrome). It participates in vesicle transport and is a negative regulator of sonic hedgehog signaling pathway, and plays different roles in different tumors. The subject group has found Rab23 in skin squamous cell carcinoma. High expression, but there is no study of the role and mechanism of Rab23 in the invasion of skin squamous cell carcinoma. This study detected the expression of Rab23 in light keratosis, abalone disease, skin squamous cell carcinoma and normal skin by immunohistochemistry, analyzed the correlation with the invasion of squamous cell carcinoma, detected the expression of Rab23 in the squamous cell carcinoma cell lines, and studied Rab23 for squamous cell carcinoma. The effect of cell invasion and preliminary discussion of the mechanism.
Experimental methods:
(1) immunohistochemistry was used to detect the expression of Rab23 in light keratosis, abalone disease, skin squamous cell carcinoma and normal skin. The difference of expression of Rab23 in each group was statistically analyzed, and the correlation between the positive expression of Rab23 and the clinicopathological features of skin squamous cell carcinoma; and Western blotting was used to detect four squamous cell carcinoma cell lines and H in HSC-2, HSQ-89, Sa-3, Tca. The expression in ACAT; (3) the effect of Rab23 on the invasiveness of squamous cell carcinoma cells by Transwell; (4) the effect of Rab23 on the migration ability of squamous cell carcinoma cells; (5) Real time RT-PCR detection of interference or overexpression of Rab23 in squamous cell carcinoma cells, MMP2, MMP9, Hedgehog signaling pathway The expression of invasion related molecule Rac1 was detected by Western blotting.
Experimental results:
(1) the expression of Rab23 in skin squamous cell carcinoma, ray keratosis, bauwen disease and normal skin: Rab23 is expressed in light keratosis, bauwen disease and skin squamous cell carcinoma, and is not expressed in normal skin, unexposed parts and medium, and low differentiated squamous cell carcinoma is a risk factor for Rab23 positive expression.
(2) the expression of Rab23 in four squamous cell carcinoma cell lines and Hacat in HSC-2, HSQ-89, Sa-3, Tca: Rab23 is expressed in four squamous cell carcinoma cell lines, expressed in HSC-2, HSQ-89 cells, low in Sa-3 and Tca, and not expressed in Hacat.
(3) the effect of Rab23 on the invasive ability of squamous cell carcinoma cells in Transwell cells: the invasion ability of HSQ-89 cells was weakened after Rab23 specific siRNA interference, and the invasion ability of Sa-3 cells was enhanced after transfection of Rab23.
The effect of Rab23 on the migration ability of squamous cell carcinoma cells was detected by cell scratch test: after siRNA interference in HSQ-89 cells, only 1 cell diameter distances were migrated between the interference group and the control group. After the transfection of Sa-3 cells, there was no statistical significance between the over expression group and the control group.
Real time RT-PCR detected the level of related molecules after interference or overexpression of Rab23: the Rab23 interference group of HSQ-89 cells MMP2, MMP9, Gli2 was lower than the control group, Ptch1, and the level of Gli1 was higher than that of the control group;
(6) Western blotting detected the expression of Rac1 after interference or overexpression of Rab23: after HSQ-89 cells interfered with Rab23 expression, the level of Rac1 was lower than that of the control group, and the level of Rac1 was higher than that of the control group after the expression of Rab23 in Sa-3 cells.
The main conclusions are as follows:
(1) Rab23 is positive in light keratosis, bauwen disease and skin squamous cell carcinoma, but not in normal skin. Rab23 is expressed in squamous cell carcinoma cell lines and Hacat is not expressed, indicating that it participates in the development of squamous cell carcinoma of the skin.
(2) Rab23 promotes the invasion of squamous cell carcinoma and does not affect cell migration.
(3) Rab23 promotes the invasion of squamous cell carcinoma possibly through Rac1, MMP2 and MMP9, probably not through the hedgehog signaling pathway.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R739.5

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