本文選題：全反式維甲酸 + 食管癌細(xì)胞��； 參考：《鄭州大學(xué)》2015年碩士論文

【摘要】：背景食管癌是發(fā)病率較高、預(yù)后較差的實(shí)體腫瘤之一。腫瘤直徑小于1mm時(shí),可以通過自身滲透作用為之提供營養(yǎng),當(dāng)腫瘤直徑達(dá)到1-2mm時(shí),腫瘤內(nèi)部發(fā)生了血管的新生現(xiàn)象,可以為腫瘤的生長提供充足的營養(yǎng),新生血管可以促進(jìn)腫瘤的發(fā)展、浸潤和轉(zhuǎn)移。多種生長因子共同參與調(diào)節(jié)著腫瘤血管的生成。其中我們研究最為廣泛和深入的生長因子是血管內(nèi)皮生長因子(VEGF),主要調(diào)控著腫瘤血管的生成,促進(jìn)了多種惡性腫瘤的生長、轉(zhuǎn)移[1-3]。另一類較大的生長因子家族是成纖維生長因子(FGF)家族,其中b FGF是第一個(gè)被鑒定的促血管生成因子[4],在血管生成中也起著重要的調(diào)節(jié)作用。誘導(dǎo)分化概念的提出,為腫瘤的治療提供了一種新的、有效的治療理念。越來越多的腫瘤專家開始關(guān)注誘導(dǎo)分化治療的研究進(jìn)展。全反式維甲酸(ATRA)作為一種最重要的誘導(dǎo)分化劑,已經(jīng)在白血病及部分實(shí)體腫瘤中得到應(yīng)用。本研究通過體外實(shí)驗(yàn)觀察ATRA對(duì)食管癌細(xì)胞系Eca109中VEGF、b FGF蛋白和m RNA表達(dá)的影響,為ATRA在食管癌抗血管生成的治療提供理論依據(jù)。研究不同濃度ATRA對(duì)食管癌細(xì)胞系Eca109 VEGF和b FGF表達(dá)的影響。方法終濃度分別為0、1、5、10μmol/L的ATRA作用于人食管癌Eca109細(xì)胞,采用噻唑藍(lán)比色法(MTT)評(píng)價(jià)藥物對(duì)細(xì)胞的增殖抑制情況;通過劃痕實(shí)驗(yàn)測定ATRA作用于Eca109 48h前后的遷移能力;采用細(xì)胞免疫組織化學(xué)法和逆轉(zhuǎn)錄酶聚合鏈?zhǔn)椒磻?yīng)(RT-PCR)分別檢測不同濃度ATRA作用于Eca109 48h后VEGF、b FGF蛋白和m RNA的表達(dá)情況。結(jié)果1.MTT結(jié)果:作用于24h后,實(shí)驗(yàn)組,即1μmol/L、5μmol/L、10μmol/L組的增殖抑制率分別為:(44.57±1.36)%、(50.26±1.64)%、(54.24±1.39)%;48h后,上述實(shí)驗(yàn)組的增殖抑制率分別為:(54.29±0.15)%、(59.52±0.74)%、(65.45±4.33)%;72h后,上述實(shí)驗(yàn)組的增殖抑制率分別為:(66.39±0.87)%、(74.33±2.69)%、(85.24±1.49)%。而對(duì)照組,0μmol/L組三個(gè)時(shí)間點(diǎn)的抑制率均為0.00%。經(jīng)統(tǒng)計(jì)學(xué)分析,各個(gè)實(shí)驗(yàn)組分別與對(duì)照組相比,差異均具有統(tǒng)計(jì)學(xué)意義(P0.05),且實(shí)驗(yàn)組之間進(jìn)行兩兩比較,差異也具有統(tǒng)計(jì)學(xué)意義(P0.05)。2.劃痕實(shí)驗(yàn)結(jié)果:1、5、10μmol/L ATRA作用于Eca109 48h后,細(xì)胞的遷移率分別為(50.82±1.61)%,(46.80±5.12)%,(32.31±3.25)%,對(duì)照組(68.81±2.64)%。0h各組細(xì)胞的遷移率均為0.00%。實(shí)驗(yàn)組數(shù)據(jù)分別與對(duì)照組相比,差異均具有統(tǒng)計(jì)學(xué)意義(P0.05),且實(shí)驗(yàn)組之間兩兩比較,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。3.免疫組化結(jié)果:VEGF蛋白陽性表達(dá)體現(xiàn)為細(xì)胞內(nèi)出現(xiàn)的棕黃或棕褐色顆粒狀,主要分布于細(xì)胞質(zhì),核膜亦可見陽性表達(dá)。b FGF蛋白陽性表達(dá)為細(xì)胞核內(nèi)被染成棕黃色,并且胞漿、核膜可見陽性表達(dá)。0、1、5、10μmol/L ATRA作用于Eca109 48h后,細(xì)胞內(nèi)VEGF蛋白相對(duì)表達(dá)量依次為:(85.43±0.53)×10-2、(77.45±0.51)×10-2、(51.43±0.34)×10-2、(21.74±0.11)×10-2;b FGF蛋白的相對(duì)表達(dá)量依次為:(71.41±0.52)×10-2、(51.63±0.46)×10-2、(38.44±0.32)×10-2、(11.24±0.21)×10-2�？梢钥闯�,隨著藥物濃度的增加,各個(gè)指標(biāo)的相對(duì)表達(dá)量逐漸減少,經(jīng)統(tǒng)計(jì)學(xué)分析,各個(gè)實(shí)驗(yàn)組分別與對(duì)照組比較,差異均具有統(tǒng)計(jì)學(xué)意義(P0.05),且實(shí)驗(yàn)組之間進(jìn)行兩兩比較,差異也具有統(tǒng)計(jì)學(xué)意義(P0.05)。4.PCR結(jié)果:0、1、5、10μmol/L ATRA作用于Eca109細(xì)胞48h后,VEGF m RNA表達(dá)量依次為:(12.40±1.11)×10-2、(7.27±0.71)×10-2、(4.63±0.45)×10-2、(1.62±0.25)×10-2;b FGF m RNA表達(dá)量依次為:(17.96±0.46)×10-2、(15.32±0.33)×10-2、(12.04±0.50)×10-2、(9.13±0.30)×10-2�？梢钥闯�,隨著藥物濃度的增加,各個(gè)指標(biāo)的表達(dá)量逐漸減少,經(jīng)統(tǒng)計(jì)學(xué)分析,各個(gè)實(shí)驗(yàn)組分別與對(duì)照組比較,差異均具有統(tǒng)計(jì)學(xué)意義(P0.05),且實(shí)驗(yàn)組之間進(jìn)行兩兩比較,差異也具有統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論1、在一定濃度范圍內(nèi),ATRA對(duì)食管癌Eca109細(xì)胞的增殖具有抑制作用,并且隨著劑量的增大或作用時(shí)間的延長,抑制作用越明顯,因此具有劑量和時(shí)間依賴性。2、在一定濃度范圍內(nèi),ATRA可以抑制Eca109細(xì)胞的遷移,抑制VEGF、b FGF的m RNA和蛋白的表達(dá),并且隨著藥物濃度的增加,抑制作用越明顯。3、ATRA可能通過下調(diào)VEGF、b FGF基因的表達(dá)影響食管癌Eca109血管新生從而抑制其生長。
[Abstract]:Background esophageal cancer is one of the solid tumors with high incidence and poor prognosis. When the diameter of the tumor is less than 1mm, it can provide nutrition through its own osmosis. When the diameter of the tumor reaches 1-2mm, the neoplasm occurs in the tumor, which can provide sufficient nutrition for the growth of the tumor. The new blood vessels can promote the development of the tumor. Spread, infiltration and metastasis. Multiple growth factors are involved in regulating the formation of tumor vessels. We study the most extensive and deep growth factor is vascular endothelial growth factor (VEGF), which mainly regulates the formation of tumor vessels, promotes the growth of many malignant tumors, and transfers the other large growth factor family of [1-3].. The FGF family, in which B FGF is the first identified angiogenic factor [4], also plays an important regulatory role in angiogenesis. The concept of induction of differentiation provides a new and effective treatment concept for the treatment of tumors. More and more cancer experts have begun to pay attention to the study of induction of differentiation therapy. All trans retinoic acid (ATRA), as one of the most important inducer and differentiation agents, has been used in leukemia and some solid tumors. In this study, the effect of ATRA on the expression of VEGF, B FGF protein and m RNA in Eca109 of esophageal cancer cell line was observed in vitro. The study provided a theoretical basis for the treatment of anti angiogenesis in gastric cancer. The effects of different concentrations of ATRA on the expression of Eca109 VEGF and B FGF in esophageal carcinoma cell lines. The final concentration of 0,1,5,10 mu mol/L was respectively on the Eca109 cells of human esophageal cancer. The proliferation inhibition of the cells was evaluated by thiazolium colorimetric assay (MTT), and the migration ability of ATRA before and after the Eca109 was measured by scratch test. Cell immuno histochemical method and reverse transcriptase polymerized chain reaction (RT-PCR) were used to detect the expression of VEGF, B FGF protein and m RNA after Eca109 48h, respectively. Results 1.MTT results: after 24h, the experimental group, namely, 1 micron mol/L, 5 micron, and 10 micron groups were (44.57 + 1.36)%, (50.26 + 1.64)%, (54.24). After 48h, the proliferation inhibition rate of the experimental group was: (54.29 + 0.15)%, (59.52 + 0.74)% and (65.45 + 4.33)%. After 72h, the inhibition rate of the experimental group was (66.39 + 0.87)%, (74.33 + 2.69)%, (85.24 + 1.49)%, and the control group was statistically analyzed by 0.00%. by statistical analysis, and the experimental groups were all divided by the control group. Compared with the control group, the differences were statistically significant (P0.05), and the difference between the 22 experimental groups was also statistically significant (P0.05).2. scratch test results: 1,5,10 mu mol/L ATRA after Eca109 48h, the cell migration rate was (50.82 + 1.61)%, (46.80 + 5.12)%, (32.31 + 3.25)%, and the control group (68.81 + 2.64)%.0h each. The migration rates of the group cells were both 0.00%. experimental group and the control group, the difference was statistically significant (P0.05), and the difference between the experimental groups was 22, and the difference was statistically significant (P0.05).3. immunohistochemical results: the positive expression of VEGF protein was manifested as brown or brown granules in the cell, mainly distributed in the cytoplasm. The positive expression of the positive expression of.B FGF protein was found to be brown and yellow in the nucleus, and the cytoplasm, and the positive expression of.0,1,5,10 mu mol/L ATRA in the nuclear membrane, was found to be (85.43 + 0.53) * 10-2, (77.45 + 0.51) * 10-2, (51.43 + 0.34) x 10-2, (21.74 + 0.11) x 10-2, B FGF eggs The relative expression of white was as follows: (71.41 + 0.52) x 10-2, (51.63 + 0.46) x 10-2, (38.44 + 0.32) x 10-2 and (11.24 + 0.21) x 10-2. can be seen that the relative expression of each index gradually decreased with the increase of drug concentration. The differences were statistically significant (P0.05), and the difference was statistically significant (P0.05). The 22 comparison between the groups was statistically significant (P0.05).4.PCR results: after 0,1,5,10 mu mol/L ATRA acted on Eca109 cell 48h, the VEGF m RNA expression was (12.40 + 1.11) x 10-2, (7.27 + 0.71) x 10-2, (4.63 + 0.45) * 10-2, (1.62 + 0.25) * 10-2, B FGF X 10-2, (12.04 + 0.50) x 10-2, (9.13 + 0.30) x 10-2. can see that with the increase of drug concentration, the expression of each index gradually decreased. By statistical analysis, the differences were statistically significant (P0.05) compared with the control group (P0.05). The difference was statistically significant (P0.05). 1, in a certain concentration range, ATRA can inhibit the proliferation of Eca109 cells in esophageal cancer, and the inhibition effect is more obvious with the increase of dose or the prolongation of action time. Therefore, it has a dose and time dependent.2. In a certain concentration range, ATRA can inhibit the migration of Eca109 cells and inhibit VEGF, m RNA and protein of B FGF. With the increase of drug concentration, the more obvious the inhibitory effect is.3, and ATRA may inhibit the growth of esophageal cancer Eca109 angiogenesis by down regulation of VEGF and the expression of B FGF gene.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R735.1

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