發(fā)布時(shí)間：2018-06-25 13:10

  本文選題：HDGF + 子宮內(nèi)膜癌��； 參考：《南方醫(yī)科大學(xué)》2014年碩士論文

【摘要】：研究背景與目的 子宮內(nèi)膜癌是婦科常見(jiàn)的惡性腫瘤之一,且隨著經(jīng)濟(jì)社會(huì)發(fā)展,發(fā)病率在全世界范圍內(nèi)呈上升趨勢(shì)。與其他腺癌的發(fā)展一樣,子宮內(nèi)膜癌的發(fā)展經(jīng)過(guò)一系列復(fù)雜變化,包括從不典型增生到高分化癌。在臨床上,年齡、肥胖、不孕、長(zhǎng)期高雌激素水平等都可能成為子宮內(nèi)膜癌發(fā)生的危險(xiǎn)因素,而長(zhǎng)期暴露于高雌激素水平被認(rèn)為是最危險(xiǎn)的因素。子宮內(nèi)膜癌的分類方法很多種,目前根據(jù)臨床癥狀及病理類型把子宮內(nèi)膜癌分為兩種類型,即Ⅰ型和Ⅱ型。第一種類型,Ⅰ型子宮內(nèi)膜癌即雌激素依賴型子宮內(nèi)膜癌,常發(fā)生于圍絕經(jīng)期婦女,尤其是子宮內(nèi)膜長(zhǎng)期處于高雌激素水平刺激而無(wú)孕激素抵抗的婦女,在白人婦女尤為常見(jiàn)。這種類型的子宮內(nèi)膜癌病理類型類似正常子宮內(nèi)膜。Ⅱ型子宮內(nèi)膜癌,即非雌激素依賴型,發(fā)病與雌激素?zé)o明顯關(guān)系。此類型常發(fā)生于老年、絕經(jīng)后的婦女,常見(jiàn)于非裔美國(guó)人。這類子宮內(nèi)膜癌的病理形態(tài)屬少見(jiàn)類型,如子宮內(nèi)膜漿液性乳頭癌、透明細(xì)胞癌等,且腫瘤分化差,惡性程度高,常侵潤(rùn)到子宮肌層深部,預(yù)后不良。 子宮內(nèi)膜癌的發(fā)生、發(fā)展是多種因素參與、多步驟的復(fù)雜過(guò)程,多數(shù)學(xué)者認(rèn)為是下列因素相互作用的結(jié)果：激素的調(diào)節(jié),基因突變,粘附分子,細(xì)胞凋亡等。若子宮內(nèi)膜癌患者在早期診斷并經(jīng)正規(guī)治療,5年生存率為80%。但不是所有患者能在早期階段發(fā)現(xiàn),因此,轉(zhuǎn)移是子宮內(nèi)膜癌患者死亡的主要原因。然而,子宮內(nèi)膜癌發(fā)生發(fā)展的分子機(jī)制尚不明確。 肝癌衍生生長(zhǎng)因子(Hepatoma-derived growth factor,HDGF)是一種肝素結(jié)合的生長(zhǎng)因子,最初從人肝癌HuH-7細(xì)胞系無(wú)血清培養(yǎng)的上清液中分離得到。HDGF在胚胎組織表達(dá)最高,且與多器官如心血管系統(tǒng)、肝臟及腎臟等器官發(fā)育密切相關(guān)。HDGF在血管增生、內(nèi)皮細(xì)胞增殖、遷移等細(xì)胞活動(dòng)中發(fā)揮著重要作用。最開始認(rèn)為HDGF定位于包漿,但后來(lái)證實(shí)是一個(gè)含有典型的雙向核定位序列的核定位蛋白,它通過(guò)核定位序列轉(zhuǎn)移到細(xì)胞核內(nèi)而發(fā)揮維持細(xì)胞活性的功能。最近有研究發(fā)現(xiàn),有在幾種小鼠和人的癌中HDGF的表達(dá)高于癌旁組織。還有研究發(fā)現(xiàn),HDGF表達(dá)與腫瘤的惡性生長(zhǎng)有關(guān),如增殖、侵襲及轉(zhuǎn)移。因此,HDGF可能是一個(gè)與腫瘤預(yù)后相關(guān)的因子。 磷脂酰肌醇3一激酶(PI3Ks),因具有通過(guò)磷酸化肌醇環(huán)的3'-OH基團(tuán)的肌醇磷脂,以產(chǎn)生第二信使磷脂酰肌醇-3,4,5-三磷酸酯的能力(PI-3,4,5-P(3))而構(gòu)成脂質(zhì)激酶家族。包漿膜上的PI3K通過(guò)激活RPTK產(chǎn)生PI(3,4,5)P(3)和PI(3,4)P(2),這些脂質(zhì)與AKT結(jié)合后轉(zhuǎn)移到包膜內(nèi)側(cè),磷酸化后并被PDKl和PDK2激活�；罨腁kt調(diào)節(jié)涉及細(xì)胞存活,細(xì)胞周期和細(xì)胞生長(zhǎng)的調(diào)節(jié)等眾多功能。近年來(lái)國(guó)內(nèi)外研究發(fā)現(xiàn),很多腫瘤的發(fā)生發(fā)展與PI3K/AKT信號(hào)通路的異常激活相關(guān)。PI3K和Akt活性的異常增高與腫瘤細(xì)胞的血管形成、侵襲轉(zhuǎn)移、抗凋亡、異常增殖等生物學(xué)行為密切相關(guān)。 目前,尚無(wú)有關(guān)HDGF與子宮內(nèi)膜癌的文獻(xiàn)報(bào)道。本課題的研究目的為明確HDGF基因在子宮內(nèi)膜癌中的功能,探討其與臨床參數(shù)的關(guān)系及參與的信號(hào)轉(zhuǎn)導(dǎo)通路,為子宮內(nèi)膜癌發(fā)生發(fā)展的分子機(jī)制研究提供新思路。 研究?jī)?nèi)容與方法 1.HDGF基因在子宮內(nèi)膜癌組織中表達(dá)及與臨床參數(shù)之間的關(guān)系 (1)本研究所采用的122例子宮內(nèi)膜癌、20例正常子宮內(nèi)膜及22例不典型增生內(nèi)膜組織切片均來(lái)自于廣州醫(yī)科大學(xué)(原廣州醫(yī)學(xué)院)第三附屬醫(yī)院2002-2008年間住院患者。子宮內(nèi)膜癌患者在手術(shù)前均未接受化療或放療�；颊咂骄挲g51.3歲(30-82歲)。臨床隨訪時(shí)間是5至96月。所有標(biāo)本均經(jīng)病理檢查確診,術(shù)后根據(jù)FIGO2009對(duì)患者進(jìn)行手術(shù)-病理分期。 (2)采用SP免疫組織化學(xué)方法檢測(cè)122例子宮內(nèi)膜癌、22例子宮內(nèi)膜不典型增生、20例正常子宮內(nèi)膜組織的HDGF表達(dá)情況,并分析HDGF的表達(dá)與臨床參數(shù)之間的關(guān)系。 (3)由兩位病理專家對(duì)所有免疫組化染色的切片進(jìn)行雙盲分析。HDGF蛋白表達(dá)定位于胞核,按陽(yáng)性細(xì)胞個(gè)數(shù)占比例計(jì)算,其中根據(jù)陽(yáng)性細(xì)胞個(gè)數(shù)比例≥80%為高表達(dá),80%為低表達(dá)。 2.HDGF基因在子宮內(nèi)膜癌中功能初步研究 (1)構(gòu)建靶向HDGF的shRNA慢病毒干擾載體,包裝成成熟顆粒,感染高成瘤細(xì)胞,建立穩(wěn)定干擾HDGF的子宮內(nèi)膜癌細(xì)胞株,以空載作為對(duì)照。 (2)以空載作為對(duì)照,利用MTT法檢測(cè)干擾HDGF的子宮內(nèi)膜癌細(xì)胞的增殖能力。 (3)以空載作為對(duì)照,利用平板克隆法檢測(cè)干擾HDGF的子宮內(nèi)膜癌細(xì)胞的生長(zhǎng)能力。 (4)以空載作為對(duì)照,Transwell小室遷移實(shí)驗(yàn)檢測(cè)干擾HDGF的子宮內(nèi)膜癌細(xì)胞的遷移能力。 (5)以空載作為對(duì)照,Boyden小室侵襲實(shí)驗(yàn)檢測(cè)干擾HDGF的子宮內(nèi)膜癌細(xì)胞的侵襲能力。 (6)檢測(cè)穩(wěn)定干擾HDGF的子宮內(nèi)膜癌細(xì)胞裸鼠皮下成瘤能力的改變。 3.HDGF基因介導(dǎo)的分子基礎(chǔ)初步研究 (1)利用western blot檢測(cè)干擾HDGF后PI3K/Akt信號(hào)通路相關(guān)基因PI3K、pPBK、pAkt、Akt的變化。 (2)Western blot檢測(cè)細(xì)胞周期相關(guān)基因CDK4、CCND1、P15、P21及EMT標(biāo)志物N-cadherin、Vimentin的表達(dá)。 4.統(tǒng)計(jì)分析 所有數(shù)據(jù)均用SPSS17.0統(tǒng)計(jì)軟件處理,當(dāng)P0.05時(shí)認(rèn)為差異具有統(tǒng)計(jì)學(xué)意義。 結(jié)果 1.HDGF基因在子宮內(nèi)膜癌組織中表達(dá)及與臨床參數(shù)之間的關(guān)系 (1)應(yīng)用免疫組織化學(xué)染色法檢測(cè)122例子宮內(nèi)膜癌石蠟切片,22例不典型增生內(nèi)膜及20例正常子宮內(nèi)膜石蠟切片的HDGF蛋白在細(xì)胞中的表達(dá)位置及過(guò)表達(dá)率。HDGF在EC組織及非腫瘤組織中均表達(dá)于胞核,在子宮內(nèi)膜癌,不典型增生內(nèi)膜及正常子宮內(nèi)膜的高表達(dá)率分別為25.5%(31/122),13.6%(3/122),5%(1/20),差別有顯著性(χ2=115.03,P0.001),子宮內(nèi)膜癌組織中HDGF的表達(dá)顯著高于非腫瘤組織。 (2)分析HDGF表達(dá)水平與子宮內(nèi)膜癌患者臨床病理參數(shù)的關(guān)系。HDGF高表達(dá)在年齡、月經(jīng)狀態(tài)、組織分型、淋巴轉(zhuǎn)移以及肌層浸潤(rùn)的深度中無(wú)統(tǒng)計(jì)學(xué)意義,在FIGO分期差異有顯著意義(P=0.032)。 (3)我們用Kaplan-Meier法及l(fā)og-rank檢驗(yàn)進(jìn)行生存率的估計(jì)與生存曲線的分析,在122例子宮內(nèi)膜癌患者組織中,發(fā)現(xiàn)HDGF的陽(yáng)性表達(dá)與患者生存率呈負(fù)相關(guān)(P=0.001)。多因素分析顯示,HDGF不是EC患者預(yù)后的獨(dú)立影響因素(P=0.111)。 2.HDGF基因在子宮內(nèi)膜癌細(xì)胞功能的初步研究 (1)抑制HDGF表達(dá)對(duì)子宮內(nèi)膜癌細(xì)胞生物學(xué)特性的影響。構(gòu)建靶向HDGF的ShRNA1慢病毒干擾載體,穩(wěn)定干擾子宮內(nèi)膜癌細(xì)胞HEC-1B和Ishikawa,由于感染效率達(dá)到90%以上,western blot檢測(cè)干擾后HDGF蛋白的表達(dá)明顯下降,可直接用于后續(xù)實(shí)驗(yàn)。陽(yáng)性干擾多克隆命名為HEC-1B shHDGF、Ishikawa shHDGF,陰性對(duì)照多克隆為HEC-1B PLV-Ctr、Ishikawa PLV-Ctr. (2)MTT法檢測(cè)細(xì)胞增殖情況,與空載對(duì)照細(xì)胞HEC-1B PLV-Ctr、Ishikawa PLV-Ctr相比,干擾細(xì)胞HEC-1B shHDGF、Ishikawa shHDGF生長(zhǎng)速度明顯減慢,差異有統(tǒng)計(jì)學(xué)意義(HEC-1B細(xì)胞株F=259.5, P0.001;Ishikawa細(xì)胞株F=38.324,P0.001) (3)平板克隆法檢測(cè)細(xì)胞生長(zhǎng)情況,與空載對(duì)照細(xì)胞HEC-1B PLV-Ctr、Ishikawa PLV-Ctr相比,干擾細(xì)胞HEC-1B shHDGF、Ishikawa shHDGF生長(zhǎng)速度明顯減慢,差異有統(tǒng)計(jì)學(xué)意義(HEC-1B細(xì)胞株t=3.74, P=0.02;Ishikawa細(xì)胞株t=4.9,P=0.008)。 (4)Transwell小室遷移實(shí)驗(yàn),細(xì)胞培養(yǎng)12h后HEC-1B shHDGF、Ishikawa shHDGF穿過(guò)聚碳酸酯膜的數(shù)量少于HEC-1B PLV-Ctr、Ishikawa PLV-Ctr (HEC-IB細(xì)胞株t=7.17,P=0.002;Ishikawa細(xì)胞株t=7.89,P0.001)。提示干擾HDGF后,細(xì)胞遷移能力減弱。 (5)Boyden小室侵襲實(shí)驗(yàn),與空載細(xì)胞相比,HEC-1B shHDGF、Ishikawa shHDGF穿過(guò)聚碳酸酯膜的數(shù)量明顯減少,差異具有統(tǒng)計(jì)學(xué)意義(HEC-1B細(xì)胞株t=9.04,P0.001;Ishikawa細(xì)胞株t=8.48,P0.001),提示干擾HDGF后,細(xì)胞侵襲能力減弱。 (6)裸鼠皮下成瘤,與空載對(duì)照組細(xì)胞相比,HEC-1B shHDGF、Ishikawa shHDGF兩處理組細(xì)胞的裸鼠成瘤速度顯著減慢,差異有統(tǒng)計(jì)學(xué)意義(HEC-1BF=255.9,P0.001;Ishikawa F=216.5,P0.001)。 3. HDGF在子宮內(nèi)膜癌中介導(dǎo)的分子機(jī)制研究 (1)采用western blot檢測(cè)了干擾HDGF后PI3K/Akt信號(hào)通路相關(guān)基因P13K、pPI3K、pAkt、Akt的表達(dá)。結(jié)果顯示,與空載對(duì)照組細(xì)胞相比,HEC-1B shHDGF、 Ishikawa shHDGF組的PI3K、Akt無(wú)明顯變化,pPI3K、pAkt表達(dá)下調(diào)。 (2)干擾HDGF后細(xì)胞周期相關(guān)基因及EMT標(biāo)志物的表達(dá)變化。結(jié)果顯示,與空載對(duì)照組細(xì)胞相比,HEC-1B shHDGF、Ishikawa shHDGF組的P15、P21在干擾細(xì)胞中表達(dá)上調(diào),CCND1、CDK4表達(dá)下降。EMT相關(guān)N-cadherin、 Vimentin表達(dá)下調(diào)。 結(jié)論 1.HDGF蛋白在子宮內(nèi)膜癌的核高表達(dá)率明顯高于子宮內(nèi)膜不典型增生及正常子宮內(nèi)膜組織,且HDGF的核高表達(dá)率與子宮內(nèi)膜癌病人的FIGO分期正相關(guān),與病人的生存時(shí)間負(fù)相關(guān)。因此,HDGF很可能是一個(gè)子宮內(nèi)膜癌預(yù)后不良的影響因素。 2.干擾HDGF后,子宮內(nèi)膜癌細(xì)胞的增殖、遷移、侵襲能力顯著減弱,進(jìn)一步提示HDGF在子宮內(nèi)膜癌中發(fā)揮了癌基因的功能。 3. HDGF基因通過(guò)介導(dǎo)PI3K/AKT信號(hào)通路影響子宮內(nèi)膜癌的發(fā)生發(fā)展。
[Abstract]:Research background and purpose
Endometrial carcinoma is one of the most common malignant tumors in gynecology, and with the development of economy and society, the incidence of endometrium is rising worldwide. As with the development of other adenocarcinoma, the development of endometrial cancer has undergone a series of complex changes, including untypical hyperplasia to highly differentiated carcinoma. In clinical, age, obesity, infertility, long term high female irritable disease Hormone levels may be a risk factor for endometrial carcinogenesis, and long-term exposure to high estrogen levels is considered as the most dangerous factor. Endometrial carcinoma is classified into two types according to clinical symptoms and pathological types, i. e. type I and type II. Type I, type I uteri Membrane cancer is estrogen dependent endometrial cancer, often occurring in perimenopausal women, especially in women who have long estrogen levels in the endometrium without progestin resistance, especially in white women. This type of endometrial carcinoma is similar to normal endometrium. Type II endometrial carcinoma, that is, non estrogen This type is often found in elderly, postmenopausal women and common in African Americans. This type of endometrial carcinoma is a rare type of endometrial carcinoma, such as serous papillary carcinoma of the endometrium, clear cell carcinoma and so on, and the tumor is poorly differentiated and has a high degree of evil, often embellish into the deep myometrium of the uterus and has poor prognosis.
The development of endometrial carcinoma is a complex process of multiple factors and multiple steps. Most scholars believe that the following factors interact: hormone regulation, gene mutation, adhesion molecules, cell apoptosis and so on. If endometrial cancer patients are diagnosed early and are treated with regular therapy, the 5 year survival rate is 80%. but not all patients can be in In the early stage, metastasis is the main cause of death in endometrial cancer. However, the molecular mechanism of endometrial cancer is unclear.
Hepatoma-derived growth factor (HDGF) is a heparin binding growth factor. It was initially isolated from the serum-free supernatant of human hepatocellular carcinoma HuH-7 cell line and obtained the highest expression of.HDGF in the embryonic tissue, and it was closely related to the proliferation of multiple organs such as the cardiovascular system, the liver and kidney and other organs, and.HDGF was increased in blood vessels. Life, endothelial cell proliferation, migration and other cellular activities play an important role. First, HDGF is located in the plasma, but later proved to be a nuclear localizing protein containing a typical bi-directional nuclear location sequence, which is transferred into the nucleus by nuclear location sequence and plays the function of maintaining the cell activity. The expression of HDGF in mice and human cancers is higher than that in paracancerous tissues. Other studies have found that the expression of HDGF is associated with the malignant growth of the tumor, such as proliferation, invasion and metastasis. Therefore, HDGF may be a factor associated with the prognosis of the tumor.
The phosphatidyl inositol 3 kinase (PI3Ks) is composed of the 3'-OH group of inositol phosphorylated by the inositol phosphatidylcholine, which produces the second messenger of the phosphatidylinositol -3,4,5- three phosphate (PI-3,4,5-P (3)) and constitutes the lipid kinase family. The PI3K on the plasma membrane generates PI (3,4,5) P (3) and PI (3,4) P (2) by activating RPTK. It is transferred to the inside of the capsule and activated by PDKl and PDK2 after phosphorylation. The activation of Akt regulation involves many functions such as cell survival, cell cycle and cell growth regulation. In recent years, domestic and foreign studies have found that the occurrence and development of many tumors are associated with abnormal activation of.PI3K and Akt associated with abnormal activation of PI3K/AKT signaling pathways and tumor cells Angiogenesis, invasion and metastasis, anti apoptosis, abnormal proliferation and other biological behaviors are closely related.
At present, there is no literature about HDGF and endometrial carcinoma. The purpose of this study is to clarify the function of HDGF gene in endometrial carcinoma, to explore the relationship with the clinical parameters and the signal transduction pathway involved in the study of the molecular mechanism of the development of endometrial cancer.
Research content and method
Expression of 1.HDGF gene in endometrial carcinoma and its relationship with clinical parameters
(1) 122 cases of endometrial carcinoma, 20 cases of normal endometrium and 22 cases of atypical hyperplasia of endometrium were from 2002-2008 years of hospitalization in Third Affiliated Hospitals of Guangzhou Medical University (former Guangzhou Medical College). Patients with endometrial cancer were not treated with chemotherapy or radiotherapy before operation. The average age of the patients was 51.3 years (30-82). The clinical follow-up time was from 5 to 96 months. All the specimens were confirmed by pathological examination. Postoperative pathological stages were performed according to FIGO2009.
(2) SP immunohistochemical method was used to detect the HDGF expression of endometrial carcinoma in 122 cases, 22 cases of endometrial atypical hyperplasia and 20 cases of normal endometrium, and the relationship between the expression of HDGF and the clinical parameters was analyzed.
(3) a double blind analysis of all immunohistochemical staining by two pathological experts was used to determine the expression of.HDGF protein in the nucleus, which was calculated according to the number of positive cells, among which the number of positive cells was more than 80%, and 80% was low.
Preliminary study on the function of 2.HDGF gene in endometrial carcinoma
(1) construction of shRNA lentivirus interference carrier for target HDGF, packed into mature particles, infected high tumor cells, and established endometrial carcinoma cell lines that were stable to interfere with HDGF, and used no load as control.
(2) using no-load as control, MTT assay was used to detect the proliferation ability of endometrial cancer cells interfering with HDGF.
(3) using no-load as control, the growth ability of endometrial cancer cells interfering with HDGF was detected by plate cloning assay.
(4) with no load as a control, Transwell cell migration assay was used to detect the migration ability of HDGF endometrial cancer cells.
(5) with no load as a control, Boyden cell invasion assay was used to detect the invasion ability of HDGF endometrial cancer cells.
(6) to detect the change of subcutaneous tumor formation ability of endometrial carcinoma cells with stable HDGF interference in nude mice.
A preliminary study on the molecular basis mediated by 3.HDGF gene
(1) using Western blot to detect the changes of PI3K/Akt signaling pathway related genes PI3K, pPBK, pAkt and Akt after interfering with HDGF.
(2) Western blot was used to detect the expression of CDK4, CCND1, P15, P21 and EMT markers N-cadherin and Vimentin in cell cycle related genes.
4. statistical analysis
All data were processed by SPSS17.0 statistical software. When P0.05 was considered, the difference was statistically significant.
Result
Expression of 1.HDGF gene in endometrial carcinoma and its relationship with clinical parameters
(1) the expression and overexpression of HDGF protein in 122 cases of endometrial carcinoma, 22 cases of atypical endometrium and 20 normal endometrium paraffin sections were detected by immunohistochemical staining, and the expression rate of.HDGF was expressed in the nucleus in EC tissue and non tumor tissues, in endometrial carcinoma, atypical hyperplasia endometrium and positive. The high expression rate of normal endometrium was 25.5% (31/122), 13.6% (3/122), 5% (1/20), and the difference was significant (x 2=115.03, P0.001). The expression of HDGF in endometrial carcinoma was significantly higher than that of non tumor tissue.
(2) analysis of the relationship between the expression level of HDGF and the clinicopathological parameters of endometriosis patients, the high expression of.HDGF was not statistically significant in age, menstrual state, tissue typing, lymphatic metastasis and the depth of myometrium infiltration, and there was significant difference in FIGO staging (P=0.032).
(3) we used the Kaplan-Meier method and the log-rank test to estimate the survival rate and the survival curve. In 122 endometrium cancer patients, we found that the positive expression of HDGF was negatively correlated with the survival rate of the patients (P=0.001). Multifactor analysis showed that HDGF was not an independent influence factor (P=0.111) of EC patients.
Preliminary study on the function of 2.HDGF gene in endometrial carcinoma cells
(1) to inhibit the effect of HDGF expression on the biological characteristics of endometrial carcinoma cells. To construct the ShRNA1 lentivirus interference carrier for targeting HDGF, to stabilize the endometrial cancer cells HEC-1B and Ishikawa, because the infection efficiency reached more than 90%, and the HDGF protein was obviously decreased after the Western blot detection. The positive interference can be directly used in the follow-up experiment. The polyclonal name was HEC-1B shHDGF, Ishikawa shHDGF, negative control polyclonal HEC-1B PLV-Ctr, Ishikawa PLV-Ctr..
(2) MTT assay was used to detect cell proliferation. Compared with HEC-1B PLV-Ctr, Ishikawa PLV-Ctr, HEC-1B shHDGF and Ishikawa shHDGF growth rate were significantly slowed down, and the difference was statistically significant (HEC-1B cell strain F=259.5, P0.001; Ishikawa cell strain).
(3) cell growth was detected by flat clones. Compared with HEC-1B PLV-Ctr, Ishikawa PLV-Ctr, HEC-1B shHDGF and Ishikawa shHDGF, the growth rate of Ishikawa shHDGF was significantly slowed down, and the difference was statistically significant (t=3.74, P=0.02, Ishikawa fine cell lines).
(4) Transwell cell migration experiment, after cell culture 12h HEC-1B shHDGF, the number of Ishikawa shHDGF through polycarbonate membrane is less than HEC-1B PLV-Ctr, Ishikawa PLV-Ctr (HEC-IB cell strain t=7.17, purified cell strain).
(5) Boyden chamber invasion experiment, compared with empty cells, the number of HEC-1B shHDGF and Ishikawa shHDGF through polycarbonate membrane decreased significantly, the difference was statistically significant (HEC-1B cell line t=9.04, P0.001; Ishikawa cell line t=8.48, P0.001), suggesting that after interference HDGF, the cell invasiveness weakened.
(6) nude mice subcutaneous tumor, compared with the empty control group cells, HEC-1B shHDGF, Ishikawa shHDGF two treated group cells of the tumor growth rate of nude mice significantly slowed, the difference was statistically significant (HEC-1BF=255.9, P0.001; Ishikawa F=216.5, P0.001).
Molecular mechanism of 3. HDGF mediating in endometrial carcinoma
(1) the expression of PI3K/Akt signaling pathway related genes P13K, pPI3K, pAkt, Akt after HDGF interference was detected by Western blot. The results showed that the HEC-1B shHDGF and Ishikawa shHDGF group were not significantly changed compared with those in the empty control group.
(2) changes in the expression of cell cycle related genes and EMT markers after interference with HDGF. The results showed that the expression of P15 in HEC-1B shHDGF and Ishikawa shHDGF group was up regulation, CCND1, CDK4 expression decreased in.EMT related N-cadherin and down regulation compared with those in the empty control group.
conclusion
The high expression rate of 1.HDGF protein in endometrial carcinoma is significantly higher than that of endometrium atypical hyperplasia and normal endometrium, and the high expression of HDGF is positively related to FIGO staging in patients with endometrial cancer and is negatively related to the survival time of patients. Therefore, HDGF is likely to be an influential factor in the poor prognosis of endometrial carcinoma.
2. after interfering with HDGF, the proliferation, migration and invasion ability of endometrial cancer cells were significantly weakened, further suggesting that HDGF played an oncogene function in endometrial carcinoma.
3. HDGF gene mediates the occurrence and development of endometrial carcinoma through mediating PI3K/AKT signaling pathway.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.33

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 梁傳余,張發(fā)文,曾祥國(guó);p15蛋白在低分化鼻咽癌中的表達(dá)[J];臨床耳鼻咽喉科雜志;2002年11期

，

本文編號(hào)：2066047