本文選題：黑色素瘤 + 轉(zhuǎn)移　； 參考：《揚(yáng)州大學(xué)》2008年碩士論文

【摘要】： 目的:采用深度免疫缺陷動物,并通過手術(shù)切除腫瘤進(jìn)行高轉(zhuǎn)移模型體內(nèi)綜合篩選的實驗思路,建立鼠源和人源黑色素瘤小鼠高轉(zhuǎn)移模型及相應(yīng)細(xì)胞系,同時觀察相關(guān)生物學(xué)特性,探討轉(zhuǎn)移相關(guān)機(jī)理,為腫瘤的預(yù)防、診斷和治療等提供理想的動物模型。 方法:1.小鼠黑色素瘤高轉(zhuǎn)移模型的體內(nèi)篩選和細(xì)胞系的建立: 將小鼠黑色素瘤B16移植于T、B、NK細(xì)胞聯(lián)合免疫缺陷的BNX小鼠皮下,首先進(jìn)行皮下移植→手術(shù)切除→肺轉(zhuǎn)移→皮下移植的連續(xù)三代體內(nèi)篩選,再按照皮下移植→肺轉(zhuǎn)移→皮下移植的方法繼續(xù)進(jìn)行三代篩選,建立移植性自發(fā)高轉(zhuǎn)移模型,同時進(jìn)行腫瘤的生長、轉(zhuǎn)移情況和病理組織學(xué)的觀察。采用組織塊法對小鼠黑色素瘤高轉(zhuǎn)移腫瘤組織進(jìn)行原代培養(yǎng)及建立細(xì)胞系,觀察腫瘤細(xì)胞的形態(tài)學(xué)、生長速度、體外侵襲能力、染色體形態(tài)分析、細(xì)胞周期變化、體內(nèi)驗證皮下移植的成瘤率和轉(zhuǎn)移情況。 2.人黑色素瘤高轉(zhuǎn)移模型的體內(nèi)篩選及微轉(zhuǎn)移動態(tài)檢測: 利用人黑色素瘤A357細(xì)胞株進(jìn)行SCID小鼠皮下移植實驗時,發(fā)現(xiàn)6只動物中有1只發(fā)生明顯肺轉(zhuǎn)移,取肺轉(zhuǎn)移灶移植至SCID小鼠皮下擴(kuò)增。采用皮下移植→肺轉(zhuǎn)移→皮下移植體內(nèi)反復(fù)篩選的方法,建立皮下移植小鼠肺高轉(zhuǎn)移動物模型(血路轉(zhuǎn)移合并淋巴道轉(zhuǎn)移)及相應(yīng)的人黑色素瘤高轉(zhuǎn)移細(xì)胞系,并進(jìn)行相關(guān)生物學(xué)特性的初步研究。同時應(yīng)用該細(xì)胞系建立BNX小鼠皮下移植瘤模型,通過Alu-PCR進(jìn)行動態(tài)、定性的肺微轉(zhuǎn)移檢測。 結(jié)果:1.小鼠黑色素瘤高轉(zhuǎn)移模型的體內(nèi)篩選和細(xì)胞系的建立: 通過體內(nèi)綜合篩選,成功建立了BNX小鼠B16黑色素瘤皮下移植肺高轉(zhuǎn)移模型。該模型的皮下移植成瘤率可達(dá)100%,35d時肺轉(zhuǎn)移率達(dá)到91.7%(11/12) ,轉(zhuǎn)移程度達(dá)到+++。同時建成相應(yīng)的高轉(zhuǎn)移細(xì)胞系,將其命名為B16-sci,其倍增時間為46.07h,自然凋亡率顯著低于B16細(xì)胞(P0.05),體外侵襲能力顯著強(qiáng)于B16細(xì)胞(P0.05),染色體分析結(jié)果表明B16-sci細(xì)胞符合小鼠惡性腫瘤細(xì)胞染色體的特點(diǎn)。體內(nèi)驗證結(jié)果表明,B16-sci細(xì)胞移植于BNX小鼠皮下,已能穩(wěn)定形成100%(6/6)肺轉(zhuǎn)移,同時提高了該細(xì)胞在C57BL/6J小鼠皮下移植后的肺轉(zhuǎn)移率。 2.人黑色素瘤SCI-375高轉(zhuǎn)移模型的篩選及微轉(zhuǎn)移動態(tài)檢測: 經(jīng)體內(nèi)反復(fù)篩選,成功建立了皮下移植小鼠肺轉(zhuǎn)移動物模型(血路轉(zhuǎn)移合并淋巴道轉(zhuǎn)移)。潛伏期從篩選初期的7-14天逐漸縮短至5天左右,荷瘤壽命從60-75天下降并穩(wěn)定在45天左右。篩選傳代的243只SCID小鼠,皮下100%成瘤,肺轉(zhuǎn)移率隨著篩選的進(jìn)行,逐代提高,第6代起肺轉(zhuǎn)移率保持在100%。建成了相應(yīng)的高轉(zhuǎn)移細(xì)胞系,并將其命名為SCI-375,其倍增時間為21.60h,體外侵襲能力強(qiáng)于未篩選的A375(P0.05),染色體分析結(jié)果顯示該細(xì)胞具有人類惡性腫瘤細(xì)胞染色體的特點(diǎn)。體內(nèi)驗證結(jié)果表明:SCI-375在不同免疫缺陷動物體內(nèi)均能表達(dá)較高的轉(zhuǎn)移率, BNX小鼠肺轉(zhuǎn)移率為100%(13/13),裸小鼠肺轉(zhuǎn)移率為90.91%(10/11)。Alu-PCR結(jié)果顯示:2W時,PCR檢測和常規(guī)病理檢測肺轉(zhuǎn)移均為陰性;3W、4W、5W時,PCR檢測肺轉(zhuǎn)移率分別為(37.5%)3/8、(75%)6/8、(100%)10/10,常規(guī)病理檢測肺轉(zhuǎn)移率分別為(0%)0/8、(37.5%)3/8、(50%)5/10�？梢�,分子生物學(xué)的方法可以早期檢測腫瘤微轉(zhuǎn)移情況的發(fā)生,其靈敏度和高效性要優(yōu)于常規(guī)病理檢測。 結(jié)論:1.利用深度免疫缺陷動物以及手術(shù)切除腫瘤進(jìn)行高轉(zhuǎn)移模型的體內(nèi)篩選的實驗思路,成功建立了一個肺轉(zhuǎn)移率高、轉(zhuǎn)移特性穩(wěn)定、直觀性好、操作簡便的B16黑色素瘤皮下移植小鼠自發(fā)高轉(zhuǎn)移模型及相應(yīng)的細(xì)胞系。 2.成功建立了人黑色素瘤皮下移植小鼠自發(fā)高轉(zhuǎn)移模型及相應(yīng)的細(xì)胞系,而且存在血路和淋巴兩個轉(zhuǎn)移途徑;在不同免疫缺陷小鼠體內(nèi)表達(dá)和應(yīng)用并獲得證實。用PCR技術(shù)的高敏感性,通過擴(kuò)增特異的基因片段,對其在小鼠體內(nèi)的微轉(zhuǎn)移進(jìn)行動態(tài)檢測。 3.為探討腫瘤轉(zhuǎn)移的生物學(xué)機(jī)制和抗轉(zhuǎn)移治療提供了理想的動物模型及相應(yīng)的細(xì)胞系。
[Abstract]:Objective: to establish a high metastasis model and the corresponding cell lines in mice and human melanoma mice, and to explore the related biological characteristics and explore the mechanism of metastasis related to the prevention, diagnosis and treatment of tumor. An animal model you want.
Methods: in vivo screening and cell line establishment of 1. high metastatic melanoma models in mice:
The murine melanoma B16 was transplanted subcutaneously into the T, B and NK cells combined with the immune deficient BNX mice. First, subcutaneous transplantation, surgical resection, pulmonary metastasis and subcutaneous transplantation were screened in three successive generations. Then the three generation screening was continued according to subcutaneous transplantation, pulmonary metastasis and subcutaneous transplantation, and a spontaneous high transfer model was established. The tumor growth, metastasis and histopathological observation were carried out. Tissue block method was used to carry out primary culture and cell lines in mice with high metastatic tumor of melanoma. The morphology, growth speed, invasion ability, chromosome morphology and cell cycle change of the tumor cells were observed, and the tumor formation of subcutaneous transplantation was verified in vivo. Rate and transfer.
In vivo screening and micrometastasis dynamic detection of 2. human melanoma models with high metastasis:
When the human melanoma A357 cell line was used for subcutaneous transplantation of SCID mice, it was found that 1 of the 6 animals had obvious pulmonary metastasis, and the lung metastasis was transplanted to the SCID mice subcutaneously. The animal model of lung high metastasis (blood transfer) was established by subcutaneous transplantation, pulmonary metastasis and subcutaneous transplantation in vivo. A preliminary study on the related biological characteristics of the human melanoma high metastatic cell line and the corresponding human melanoma metastasis cell line was carried out. The cell line was used to establish the model of the subcutaneous transplantation tumor in BNX mice, and the dynamic and qualitative lung micrometastasis was detected by Alu-PCR.
Results: 1. in vivo screening and cell line establishment of mouse melanoma high metastatic model:
Through the comprehensive screening in vivo, the lung high metastasis model of subcutaneous transplantation of BNX mice with B16 melanoma was successfully established. The rate of subcutaneous transplantation of this model could reach 100%, the lung metastasis rate of 35d reached 91.7% (11/12), the degree of metastasis reached + + + + +. At the same time, the corresponding high transfer cell line was set up to be B16-sci, and its doubling time was 46.07h and natural apoptosis. The rate was significantly lower than that of B16 cells (P0.05), and the ability of invasion in vitro was significantly stronger than that of B16 cells (P0.05). The results of chromosome analysis showed that B16-sci cells were in line with the characteristics of the chromosomes of the malignant tumor cells of mice. The results showed that the transplantation of B16-sci cells to the subcutaneous of BNX mice could stabilize the formation of 100% (6/6) lung metastasis and increase the number of the cells in C57B. The lung metastasis rate of L/6J mice after subcutaneous transplantation.
Screening of SCI-375 high metastatic model of 2. human melanoma and dynamic detection of micrometastasis:
After repeated screening in the body, the animal model of lung metastases (hematogenous metastasis combined with lymphatic metastasis) was successfully established. The incubation period was shortened from 7-14 days to 5 days, and the life span of the tumor was reduced from 60-75 days to 45 days. 243 subcutaneous mice were screened, 100% subcutaneous tumors were subcutaneously, and the lung metastasis rate was selected with screening. The sixth generation of lung metastasis rate in the sixth generation maintained a corresponding high metastatic cell line and named it SCI-375, and its doubling time was 21.60h, and the invasion ability in vitro was stronger than that of unscreened A375 (P0.05). The results of chromosome analysis showed that the cell has the characteristics of the chromosome of human malignant tumor cells. The results showed that SCI-375 could express high metastasis rate in different immune deficient animals. The lung metastasis rate of BNX mice was 100% (13/13), and the lung metastasis rate of nude mice was 90.91% (10/11).Alu-PCR. The results showed that PCR detection and routine pathological examination were negative when 2W, 3W, 4W, 5W, PCR detection of lung metastasis rate was (37.5%) 3/8, (75%), (100%) 10/10, the rate of lung metastasis by routine pathological examination was (0%) 0/8, (37.5%) 3/8, (50%) 5/10., and the molecular biology method could detect the occurrence of tumor micrometastasis in early stage, and its sensitivity and efficiency were better than that of routine pathological examination.
Conclusion: 1. a high transfer rate, stable, intuitionistic and easy operation of B16 melanoma subcutaneous transplantation model and the corresponding cell lines were successfully established by using the experimental method of screening the high metastasis model of the advanced immunodeficiency animals and the surgical resection of the tumor.
2. the spontaneous high metastasis model and the corresponding cell line in the subcutaneous transplantation of human melanoma were successfully established, and there were two transfer pathways of blood and lymph, and the expression and application in different immune deficient mice were confirmed. The specific gene fragment was amplified by the Gao Min sensibility of the PCR technique, and the micrometastasis in mice was amplified by the amplification of the specific gene fragments. Dynamic testing.
3. it provides an ideal animal model and corresponding cell lines for exploring the biological mechanism of tumor metastasis and anti metastasis therapy.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R739.5;R-332

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