本文選題：膠毒霉素 + 腫瘤相關成纖維細胞　； 參考：《蘭州大學》2015年碩士論文

【摘要】：研究背景及目的：肝細胞肝癌(hepatocellular carcinoma, HCC)是最常見的原發(fā)性肝腫瘤。許多國家HCC的發(fā)病率逐年上升,死亡率居高不下。研究認為,肝臟微環(huán)境的改變已經成為HCC的病理標志之一,80%以上的HCC是由慢性肝炎-肝纖維化-肝硬化“三部曲”演變而來,同時炎癥反應和細胞外基質沉積很大程度上改變了肝臟微環(huán)境。近些年,高通量的基因組分析已經逐漸闡明了與HCC發(fā)生發(fā)展密切相關的基因網絡以及信號轉導通路,某些重要基因成為腫瘤治療藥物的靶點。但是,HCC的復發(fā)和轉移仍然困擾著我們,其原因可能與肝癌微環(huán)境相關。因此,尋找靶向微環(huán)境的藥物是我們面臨的挑戰(zhàn)。腫瘤相關成纖維細胞(tumor associated fibroblasts, TAFs)是腫瘤微環(huán)境的主要細胞組分之一,它是由肝星狀細胞和肝成纖維細胞等基質細胞演變而來,參與維持腫瘤的標志性特征。同時,TAFs可以增強腫瘤細胞的耐藥能力。既往研究發(fā)現(xiàn),膠毒霉素(Gliotoxin)可通過誘導肝星狀細胞(hepatic stellate cell, HSC)凋亡而顯著減輕肝纖維化。低濃度的Gliotoxin (0.1 μM)即可誘導HSC凋亡,而對正常肝細胞無明顯損傷,只有當濃度達到30-50 μM時方可引起正常肝細胞壞死。我們近期的實驗結果證實低濃度Gliotoxin(IC50=143.1 nM)即可顯著抑制TAFs增殖。既然Gliotoxin對肝星狀細胞(TAFs的主要來源之一)有很強的殺傷效果,我們推測Gliotoxin可能通過作用于微環(huán)境從而對HCC有治療作用。為了驗證這一推論,本研究將從以下兩方面展開：首先,分離培養(yǎng)并鑒定人HCC來源的TAFS,觀察Gliotoxin對TAFs生物學特性的影響以及凋亡誘導作用；進一步檢測Gliotoxin對TGF-β通路及其下游基因的作用；建立TAFs與肝癌細胞株的共培養(yǎng)體系,觀察Gliotoxin對共培養(yǎng)細胞的作用其次,建立DEN誘導原發(fā)性肝癌模型,研究Gliotoxin對HCC的治療作用。該研究將腫瘤微環(huán)境作為治療HCC的靶點之一,可能為臨床上HCC的治療提供新的理論依據。實驗方法：1.Gliotoxin對TAFs的作用：①分離并鑒定TAFs,將人HCC組織切成2 mm3大小,膠原酶Ⅳ消化、梯度離心后得到TAFs,進一步純化成纖維細胞。細胞免疫熒光檢測α-SMA、Vimentin、Desmin的表達。②Gliotoxin對TAFs生物學效應的影響,體外研究不同濃度Gliotoxin對TAF增殖及遷移的作用：將TAFs以3×103/孔的數(shù)量置于96孔板中,加入不同濃度的Gliotoxin培養(yǎng)6天,第6天單熒光素報告基因檢測TAFs的增殖情況,計算IC50；選定Gliotoxin合適濃度(100nM)溶于含3%胎牛血清(FBS)的培養(yǎng)基中并加入24孔板,計數(shù)5×104的TAFs接種于Transwell小室,24小時后結晶紫固定、拍照,利用ImagePro軟件計算TAFs的遷移情況。2.Gliotoxin對TAFs凋亡的誘導作用：①100nM的Gliotoxin處理TAFs后,利用Caspase3熒光試劑盒檢測不同時間點TAFs由Caspase3的表達,通過計算Caspase3活性增高倍數(shù)評價Gliotoxin對TAFs凋亡的作用。②100nM的Gliotoxin處理TAFs,4小時后收集細胞。Annexin V與碘化吡啶(PI)標記TAFs,流式細胞儀檢測凋亡情況,重點分析Gliotoxin對早期凋亡的影響。③不同濃度(0、30 nM、100n M、300 nM)的Gliotoxin處理TAFs,在不同時間點(15 min、30min、 60min、120min、180 min)檢測ATP的表達量,分析Gliotoxin對ATP的消耗能力。3.Gliotoxin對TAFs作用機制的初步探討：①TAFs培養(yǎng)基中加入100nM的Gliotoxin,48小時后提取細胞RNA,使用逆轉錄試劑盒將RNA逆轉為cDNA, Real-time PCR檢測TAFs相關標記物-SMA、Vimentin、Desmin、纖維細胞相關蛋白(FAP)以及TGF-β通路下游基因(C-myc、Smad5、AP-1、SP-1、Bcl-2)的表達。②TAFs培養(yǎng)基中分別加入0 nM、30nM、100 nM、300 nM的Gliotoxin,4小時后收集蛋白,定量后western-blot檢測TGF-β通路Smad蛋白家族的活化情況；另一組TAFs于24小時后檢測TGF-β通路報告基因3TP的熒光表達。4.Gliotoxin對TAFs-肝腫瘤細胞共培養(yǎng)系統(tǒng)的作用：①Gliotoxin處理TAFs后,收集細胞培養(yǎng)上清,同時獲取正常培養(yǎng)基、成纖維細胞株(normal fibroblasts, NFs)、TAFs培養(yǎng)上清作為條件培養(yǎng)基。將Hep3B細胞(3x103/孔)分到96孔板,加入條件培養(yǎng)基,CCK-8法檢測細胞增殖情況。②24孔板下層加入正常、NFs、TAFs、TAFs-Gliotoxin的條件培養(yǎng)基,transwell小室中加入SMMC-7721細胞(5×104),24小時后結晶紫染色,拍照、計數(shù)遷移細胞。5. Gliotoxin對DEN肝癌模型的治療作用：①Gliotoxin治療肝癌的效果評估,22周處死所有大鼠,大鼠肝臟給予門靜脈生理鹽水灌注,取出肝臟。觀察肝臟外觀的變化。取組織制作石蠟切片,切片后進行HE染色,評估Gliotoxin對肝癌的治療效果。②Gliotoxin對肝臟纖維化的影響,利用Masson和天狼星紅對肝臟組織進行染色,觀察膠原的分布情況。圖像掃描并計算膠原分布面積；免疫組織化學檢測組織中a-SMA的表達。6.統(tǒng)計學處理：數(shù)據采用SPSS 16.0統(tǒng)計軟件包進行分析,結果以X±SD表示。多組間采用One-Way ANOVA方差分析,P0.05為統(tǒng)計結果具有顯著性差異。實驗結果：Gliotoxin具有抑制TAFs的作用：①細胞免疫熒光檢測發(fā)現(xiàn)TAFs表達α-SMA、Desmin與Vimentin,其中a-SMA與vimentin在所有TAFs中均表達。而Desmin只在部分TAFs中表達。②Gliotoxin濃度低于50 nM時對細胞無明顯的殺傷效應。當Gliotoxin濃度在50-400 nM之間時,TAFs的存活率隨著藥物濃度的升高而迅速降低。根據藥敏試驗的曲線得到Gliotoxin對TAFs的IC50為147.1 nM.③ Gliotoxin可以抑制TAFs的遷移。30 nM的Gliotoxin即對TAFs的遷移具有一定的抑制作用。100 nM的Gliotoxin處理TAFs可以使TAFs的遷移數(shù)量降低至對照組的一半。當Gliotoxin濃度提升至300 nM后,遷移抑制率約為90%。④Gliotoxin促進TAFs凋亡。1)100nM的Gliotoxin處理TAFs后,Caspase3活性檢測結果發(fā)現(xiàn),15min開始Caspase3的活性已經開始增加,并且具有一定時間依賴性。180分鐘是Caspase3活性最高,為對照組的25倍以上。2)100nM的Gliotoxin處理TAFs,4小時后利用流式細胞儀檢測TAFs凋亡情況,結果顯示早期凋亡由10.83%增加至16.23%,說明Gliotoxin可以促進TAFs的凋亡。3)ATP含量測試實驗表明60分鐘時,300nM Gliotoxin處理的TAFs開始消耗ATP,此時中低濃度組的ATP含量無明顯變化。120分鐘后,100nM Gliotoxin處理TAFs的ATP含量開始下降,而300nM濃度Gliotoxin處理組ATP含量降低幅度已經超過50%。4小時后中、高濃度組的ATP持續(xù)維持在低水平。⑤Gliotoxin抑制TAFs中TGF-P通路的活化不同濃度Gliotoxin處理TAFs,4小時后收集蛋白檢測TGF-β通路活化情況,我們發(fā)現(xiàn)Smad2與Smad3表達無明顯差別,但是Smad2/3磷酸化水平顯著下降。另外,我們利用TGF-β通路報告基因檢測該通路的活化情況,發(fā)現(xiàn)Gliotoxin可以抑制TGF-β通路報告基因熒光素酶的含量。利用Real-time PCR檢測TGF-β通路下游基因表達,發(fā)現(xiàn)Gliotoxin處理后通路下游基因C-myc、Bcl-2以及AP-1表達下調。同時TAFs標記物α-SMA、Desmin、 Vimentin與FAP表達下調。⑥Gliotoxin對TAFs-肝腫瘤細胞共培養(yǎng)系統(tǒng)的作用。1)我們利用NFs、TAFs、TAFs-Gliotoxin的細胞培養(yǎng)上清處理Hep3B細胞,在不同時間點檢測腫瘤細胞的數(shù)量,結果發(fā)現(xiàn)NFs、TAFs均可促進腫瘤細胞株的增殖,但是TAFs的促增殖能力更強,如果TAFs經Gliotoxin處理,它的促增殖能力有所下降,與NFs水平相近。2)NFs促進腫瘤細胞株SMMC-7721遷移的能力較弱,TAFs則顯著增強7721細胞的遷移。Gliotoxin可以部分抑制TAFs的促遷移作用。Gliotoxin對DEN肝癌模型的治療作用：①Gliotoxin治療肝癌的效果評估。22周處死所有大鼠,取出肝臟。大體觀察各處理組肝臟的變化。發(fā)現(xiàn)模型組和溶劑對照組肝臟已經出現(xiàn)多發(fā)的肝癌結節(jié),或表面出現(xiàn)腫瘤腫塊,同時肝硬化程度較重。Gliotoxin治療組大鼠未見腫瘤腫塊。HE染色后評估Gliotoxin對肝癌的治療效果。結果顯示：模型組有6只大鼠被診斷為肝細胞癌,兩只大鼠為重度不典型增生；DMSO組5只大鼠被診斷為肝細胞癌,其余為不典型增生。肝細胞癌多數(shù)為中高分化,兩組中各有一只為低分化腫瘤；Gliotoxin治療組(0.5mg/kg與1.5mg/kg)各有1只大鼠被診斷為高分化癌,其余均為不典型增生和肝硬化。②Gliotoxin對肝臟纖維化的影響。利用Masson和天狼星紅對肝臟組織進行染色,觀察膠原的分布情況。結果顯示模型組及DMSO組膠原的表達未見明顯差異,Gliotoxin治療組特殊染色面積顯著下降。免疫組織化學檢測檢測α-SMA的表達,發(fā)現(xiàn)α-SMA在模型組與治療組的表達出現(xiàn)顯著差異,Gliotoxin治療組α-SMA表達范圍局限于匯管區(qū),而模型組α-SMA表達范圍較大,已經延伸至肝細胞所在區(qū)域。結論：1. Gliotoxin抑制TAFs的增殖、遷移,促進TAFs的凋亡。2. Gliotoxin對TAFs的抑制作用與TGF-β通路有關。3. Gliotoxin抑制實驗性肝癌的進展。
[Abstract]:Research background and objective: hepatocellular carcinoma (HCC) is the most common primary liver tumor. The incidence of HCC is rising year by year in many countries, and the mortality rate is high. It is considered that the change of liver microenvironment has become one of the pathological signs of HCC, and more than 80% of HCC is from chronic hepatitis liver fibrosis liver cirrhosis " The trilogy evolved, and the inflammatory response and extracellular matrix deposition greatly changed the liver microenvironment. In recent years, high throughput genome analysis has gradually elucidated the genetic network and signal transduction pathway closely related to the development of HCC, and some important genes have become the target of cancer therapy. But, HCC The recurrence and metastasis still perplex us, which may be related to the microenvironment of liver cancer. Therefore, the challenge to find the targeted microenvironment is the challenge we face. The tumor associated fibroblasts (TAFs) is one of the main cell components of the tumor microenvironment, which is composed of hepatic stellate cells and hepatic fibroblasts. The stromal cells evolve and participate in the maintenance of tumor markers. At the same time, TAFs can enhance the drug resistance of tumor cells. Previous studies have found that Gliotoxin can significantly reduce liver fibrosis by inducing apoptosis of hepatic stellate cell (HSC). Low concentration of Gliotoxin (0.1 u M) can induce HSC apoptosis. There is no obvious damage to normal hepatocytes. Only when the concentration reaches 30-50 M can cause normal hepatocyte necrosis. Our recent experimental results confirm that low concentration of Gliotoxin (IC50=143.1 nM) can significantly inhibit TAFs proliferation. Since Gliotoxin has a strong killing effect on hepatic stellate cells (one of the main sources of TAFs), we speculate Gl Iotoxin may have a therapeutic effect on HCC by acting on microenvironment. In order to verify this inference, this study will be carried out in the following two aspects: first, to isolate and identify the TAFS of human HCC origin, to observe the effect of Gliotoxin on the biological characteristics of TAFs and to induce apoptosis, and to further detect the Gliotoxin to TGF- beta pathway and its lower level. A co culture system of TAFs and hepatoma cells was established to observe the effect of Gliotoxin on co cultured cells, to establish a DEN induced primary liver cancer model and to study the therapeutic effect of Gliotoxin on HCC. This study could serve as one of the targets for the treatment of HCC, which may provide a new theory for the treatment of HCC in clinical practice. According to the experimental method: the effect of 1.Gliotoxin on TAFs: (1) separate and identify TAFs, cut the human HCC tissue into 2 mm3 size, digest collagenase IV, obtain TAFs after gradient centrifugation, and further purify the fibroblasts. Cell immunofluorescence detection of the expression of alpha -SMA, Vimentin, Desmin. (2) the effect of Gliotoxin on TAFs biological effect, in vitro studies are not The effect of the same concentration of Gliotoxin on the proliferation and migration of TAF: placing TAFs in 96 Kong Banzhong with the number of 3 x 103/ holes, adding different concentrations of Gliotoxin for 6 days, sixth days single fluorescein report gene detection of TAFs proliferation, IC50, selected Gliotoxin suitable concentration (100nM) in medium containing 3% fetal bovine serum (FBS) and adding 24 The hole plate, count 5 * 104 TAFs inoculated in the Transwell compartment, 24 hours later crystal violet fixed, photographed, using ImagePro software to calculate the TAFs migration of.2.Gliotoxin on the induction of TAFs apoptosis: (1) 100nM Gliotoxin treated TAFs, using Caspase3 fluorescent kit to detect the same time TAFs by Caspase3 expression, through calculation Se3 activity was increased to evaluate the effect of Gliotoxin on TAFs apoptosis. (2) 100nM Gliotoxin treated TAFs,.Annexin V and pyridine (PI) labeled TAFs were collected 4 hours later, and apoptosis was detected by flow cytometry, and the effect of Gliotoxin on early apoptosis was analyzed. At the same time point (15 min, 30min, 60min, 120min, 180 min), the expression of ATP was detected. A preliminary discussion on the action mechanism of Gliotoxin on ATP was discussed. Markers -SMA, Vimentin, Desmin, fibrous cell related protein (FAP) and the expression of downstream genes of TGF- beta pathway (C-myc, Smad5, AP-1, SP-1, Bcl-2). (2) TAFs medium added 0 nM, 100, 300, 4 hours later to collect protein and determine the activation of protein family in beta pathway; Group TAFs was tested after 24 hours to detect the effect of TGF- beta pathway reporter gene 3TP on the co culture system of TAFs- liver tumor cells: (1) after Gliotoxin treatment TAFs, collecting cell culture supernatant, obtaining normal medium, fibroblast cell line (normal fibroblasts, NFs), TAFs culture supernatant as the conditioned medium. B cells (3x103/ hole) were divided into 96 orifice plates, adding conditioned medium and CCK-8 method to detect cell proliferation. (2) the lower layer of 24 orifice was added to normal, NFs, TAFs, TAFs-Gliotoxin conditioned medium, SMMC-7721 cells (5 * 104) were added to Transwell compartment, the color of crystal violet staining after 24 hours, and counting the.5. Gliotoxin of the migratory cells to the DEN hepatoma model The effect of treatment: (1) the evaluation of the effect of Gliotoxin on liver cancer. All rats were killed 22 weeks. The liver was given to the rat liver. The liver was perfused with the portal vein. The liver was taken out. The changes of liver appearance were observed. Tissue paraffin section was made, and then HE staining was made to evaluate the effect of Gliotoxin on liver cancer. (2) the effect of Gliotoxin on liver fibrosis, and the effect of Gliotoxin on liver fibrosis The liver tissue was stained with Masson and Sirius red. The distribution of collagen was observed. The images were scanned and the area of collagen distribution was calculated. The expression of a-SMA in the immunohistochemical staining tissue was treated with.6. statistics. The data were analyzed with SPSS 16 software package, and the results were expressed in X SD. One-Way ANOVA variance scores were used among the groups. Analysis, P0.05 has significant difference for statistical results. Experimental results: Gliotoxin has the effect of inhibiting TAFs: (1) cell immunofluorescence detection found that TAFs expressed alpha -SMA, Desmin and Vimentin, in which a-SMA and vimentin were expressed in all TAFs, while Desmin was expressed only in partial TAFs. When the concentration of Gliotoxin is between 50-400 nM, the survival rate of TAFs rapidly decreases with the increase of drug concentration. According to the curve of the drug sensitivity test, the IC50 of the TAFs is 147.1 nM., and Gliotoxin can inhibit the TAFs migration.30 nM, which has a certain inhibitory effect on the migration of the.30. Toxin processing TAFs can reduce the number of migration of TAFs to half of the control group. When the Gliotoxin concentration is raised to 300 nM, the migration inhibition rate is approximately 90%. 4 Gliotoxin to promote TAFs apoptosis.1) 100nM Gliotoxin processing TAFs. Caspase3 activity was the highest in dependent.180 minutes, TAFs was treated with Gliotoxin of 100nM in the control group of more than 25 times.2). The apoptosis of TAFs was detected by flow cytometry after 4 hours. The results showed that the early apoptosis was increased from 10.83% to 16.23%, indicating that Gliotoxin could promote TAFs apoptosis.3) ATP content test experiment indicated 60 minutes. The TAFs of N treatment began to consume ATP, and at this time, the ATP content in the middle and low concentration group had no obvious change for.120 minutes, and the ATP content of TAFs in 100nM Gliotoxin treatment began to decline, while the ATP content reduction of 300nM concentration Gliotoxin treatment group was more than after the hour, and the persistence of the high concentration group remained at the low level. The activation of the pathway with different concentrations of Gliotoxin treatment TAFs, after 4 hours to collect protein to detect the activation of TGF- beta pathway, we found that there was no significant difference in the expression of Smad2 and Smad3, but the level of Smad2/3 phosphorylation was significantly decreased. Furthermore, we used TGF- beta pathway to report the activation of the pathway, and found that Gliotoxin could inhibit the TGF- beta passage. The content of luciferase in the road was reported. The downstream gene expression of TGF- beta pathway was detected by Real-time PCR. The downstream gene C-myc, Bcl-2, and AP-1 expression were down regulated after Gliotoxin treatment. Meanwhile, TAFs marker alpha -SMA, Desmin, Vimentin and FAP expression downregulated. The cell culture of NFs, TAFs and TAFs-Gliotoxin cells was used to treat Hep3B cells, and the number of tumor cells was detected at different time points. The results showed that NFs and TAFs could promote the proliferation of tumor cell lines, but TAFs was more capable of promoting proliferation. If TAFs was treated with Gliotoxin, its ability to promote proliferation was decreased, and NFs level was similar.2) NFs promoted. The migration ability of tumor cell line SMMC-7721 was weak, and TAFs enhanced 7721 cell migration.Gliotoxin significantly. The effect of.Gliotoxin on DEN liver cancer model was partly inhibited by.Gliotoxin. (1) the effect of Gliotoxin on the liver cancer was evaluated by.22 weeks to kill all rats and remove the liver. The changes of liver in each treatment group were observed in general. It was found that the liver cancer nodules were found in the liver of the model group and the solvent control group, or the tumor was found on the surface of the liver. At the same time, there was no tumor mass in the heavy.Gliotoxin group of the liver cirrhosis to evaluate the therapeutic effect of Gliotoxin on the liver cancer. The results showed that 6 rats in the model group were diagnosed as hepatocellular carcinoma and two rats were diagnosed as hepatocellular carcinoma. For the severe atypical hyperplasia, 5 rats in group DMSO were diagnosed as hepatocellular carcinoma and the rest were atypical hyperplasia. Most of the hepatocellular carcinoma were medium and high differentiated, one in each of the two groups was low differentiated tumor, and 1 rats in the Gliotoxin group (0.5mg/kg and 1.5mg/kg) were diagnosed as highly differentiated carcinoma and the others were atypical hyperplasia and cirrhosis. (2) Glioto The effect of Xin on liver fibrosis. Using Masson and Sirius red to stain the liver tissue, the distribution of collagen was observed. The results showed that the expression of collagen in the model group and the DMSO group was not significantly different. The special staining area in the Gliotoxin group decreased significantly. The expression of alpha -SMA was detected by the immunohistochemical test, and the alpha -SMA was found in the model group. The expression of Gliotoxin in the treatment group was significantly different. The expression range of alpha -SMA in the Gliotoxin treatment group was limited to the sink area, and the expression of alpha -SMA in the model group was larger and extended to the region of the liver cells. Conclusion: 1. Gliotoxin inhibits the proliferation and migration of TAFs, and promotes the inhibitory effect of TAFs apoptosis.2. Gliotoxin on TGF- beta pathway related.3. Gli. Otoxin inhibits the progress of experimental liver cancer.
【學位授予單位】：蘭州大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R735.7

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