人源性乳腺微環(huán)境小鼠模型的移植物功能研究
發(fā)布時(shí)間：2018-07-31 07:57
【摘要】：乳腺癌發(fā)病率日益增長，嚴(yán)重威脅女性生命健康。對(duì)于乳腺癌發(fā)病機(jī)制及防治的研究廣泛開展，動(dòng)物模型這一體內(nèi)研究工具在乳腺癌的研究中有著重要的作用。目前應(yīng)用較多的小鼠模型有著各自的優(yōu)缺點(diǎn)，最主要的缺陷是不能提供人源性微環(huán)境。微環(huán)境是一個(gè)由多種成分構(gòu)成的支持性網(wǎng)絡(luò)，其在腫瘤中的重要性日益得到重視。本實(shí)驗(yàn)室利用SCID（嚴(yán)重聯(lián)合免疫缺陷）小鼠先后建立了人乳腺-人乳腺及人乳腺-人骨乳腺癌原位及轉(zhuǎn)移模型，除了能更好的模擬腫瘤發(fā)生及轉(zhuǎn)移的各個(gè)過程之外，均提供了人源性組織特異性的微環(huán)境。為了對(duì)人源性系列模型的優(yōu)勢(shì)做進(jìn)一步的理解和闡釋，更為了對(duì)模型本身各項(xiàng)參數(shù)進(jìn)行定量，以便于該模型更好的被運(yùn)用，我們采用多種技術(shù)手段，對(duì)人源性移植物在小鼠體內(nèi)從適應(yīng)、恢復(fù)、正常維持至衰老的過程中，其結(jié)構(gòu)和功能的變化和適宜使用時(shí)間做了監(jiān)測(cè)和研究。 本研究中，我們按照構(gòu)建模型的標(biāo)準(zhǔn)操作將人乳腺組織植入SCID小鼠，之后每周犧牲小鼠并取出體內(nèi)移植的正常人乳腺組織，分別進(jìn)行HE染色常規(guī)病理觀察，透射電鏡觀測(cè)及Calponin、P63、Ki67、芳香化酶（Aromatase）和類固醇硫酸酯酶（steroid sulfatase, STS）的免疫組織化學(xué)染色。同時(shí)留取小鼠乳腺脂肪墊進(jìn)行了對(duì)比。另外，我們?cè)O(shè)置了“腫瘤植入組”，將新鮮人乳腺癌組織移植在SCID小鼠皮下，觀測(cè)植入8周后腫瘤組織在小鼠皮下的改變。 術(shù)后第1周小鼠植入部位的乳腺已被形成的包膜包裹，植入的組織觸感柔軟，隨植入時(shí)間的增長，植入組織體積逐漸縮小變韌。HE染色下人乳腺植入小鼠體內(nèi)后形態(tài)變化具有一定趨勢(shì)：植入最初幾周上皮細(xì)胞腫脹變形，腺體結(jié)構(gòu)模糊，這種變化在第1-3周逐漸加重，腺體及上皮細(xì)胞形態(tài)在第2-3周時(shí)達(dá)到最差，從植入第4周開始，腺體形態(tài)出現(xiàn)好轉(zhuǎn)，上皮細(xì)胞內(nèi)脂肪變性減少，細(xì)胞核形態(tài)規(guī)則，5周時(shí)腺體與植入前的腺體形態(tài)接近，此后13周，植入的腺體及上皮細(xì)胞結(jié)構(gòu)保持基本正常的腺管結(jié)構(gòu)。光鏡下植入后組織間質(zhì)始終呈現(xiàn)纖維化和玻璃樣變性，這種變性并不像腺體結(jié)構(gòu)具有隨時(shí)間的變化趨勢(shì)。 透射電子顯微鏡觀察到與普通光學(xué)顯微鏡相符的變化趨勢(shì)，結(jié)構(gòu)先變差再恢復(fù)：1周的上皮細(xì)胞細(xì)胞核形態(tài)明顯異常；2周細(xì)胞脂肪變性明顯，，細(xì)胞內(nèi)可見大量脂肪空泡，核內(nèi)也可見到脂質(zhì)包涵體，胞質(zhì)內(nèi)細(xì)胞器不明顯；3周時(shí)胞質(zhì)內(nèi)線粒體腫脹明顯；4周線粒體腫脹消退，線粒體嵴清晰；5周開始上皮細(xì)胞間隙清晰可見，且能觀測(cè)到腺上皮標(biāo)志性的橋粒連接，胞質(zhì)內(nèi)細(xì)胞器豐富，此后數(shù)周上皮細(xì)胞維持此種類似正常乳腺上皮細(xì)胞的結(jié)構(gòu)直至觀測(cè)終點(diǎn)。我們?cè)?周的標(biāo)本中觀測(cè)到了類似妊娠后期乳腺上皮表現(xiàn)的細(xì)胞，胞質(zhì)內(nèi)細(xì)胞器豐富，并且有大量小泡且向腔內(nèi)分泌，內(nèi)含兩種不同電子密度的物質(zhì)。 除大體標(biāo)本見到的包膜血管網(wǎng)之外，光鏡及電鏡均可見到血管結(jié)構(gòu)。HE切片中血管內(nèi)可見紅細(xì)胞，提示這些血管具有正常功能。 Calponin及P63是肌上皮細(xì)胞的標(biāo)志物，可提示局部乳腺微環(huán)境穩(wěn)定性。正常參照中腺管周圍有完整的肌上皮細(xì)胞包圍。但植入后1-2周的標(biāo)本中，腺管周圍Cal部分表達(dá)缺失，提示肌上皮細(xì)胞出現(xiàn)缺失，胞核染色指標(biāo)P63無明顯變化。Ki67在植入前和植入后的乳腺腺體中均呈散在表達(dá)，并未隨植入時(shí)間的推移出現(xiàn)明顯的表達(dá)增高和表達(dá)缺失的情況。局部產(chǎn)生雌激素的兩條途徑的限速酶芳香化酶（Aromatase）和類固醇硫酸酯酶（steroid sulfatase, STS）抗體在移植前及移植后各時(shí)間點(diǎn)的乳腺組織中均呈現(xiàn)染色陰性。 本實(shí)驗(yàn)選用的均是未經(jīng)交配的小鼠，故小鼠的乳腺組織始終處呈現(xiàn)靜止期乳腺的表現(xiàn)，脂肪墊內(nèi)主體為脂質(zhì)成分，中間夾雜導(dǎo)管結(jié)構(gòu)，導(dǎo)管周圍可見少量間質(zhì)細(xì)胞，切片中未見腺泡結(jié)構(gòu)。隨實(shí)驗(yàn)進(jìn)行，小鼠乳腺結(jié)構(gòu)未出現(xiàn)時(shí)間相關(guān)性改變。 腫瘤植入組共接種6只小鼠，6例成瘤均失敗，隨植入時(shí)間的增長腫塊均明顯縮小，手術(shù)取出的腫塊體積明顯小于植入前。HE染色下移植后的人乳腺癌組織周圍及內(nèi)部可見血管，血管內(nèi)可見到紅細(xì)胞。腫瘤組織周圍有包膜，腫瘤生長局限在包膜內(nèi)，部分腫瘤中心出現(xiàn)壞死區(qū)域。 正常人乳腺組織在移植到小鼠體內(nèi)后經(jīng)歷了適應(yīng)期（1-3w）—恢復(fù)期（4w）—正常結(jié)構(gòu)維持期（5w-13w）—衰老期四個(gè)階段的時(shí)間相關(guān)性變化�；謴�(fù)期后，小鼠體內(nèi)的人乳腺組織可在相當(dāng)長的時(shí)間內(nèi)維持人類正常乳腺結(jié)構(gòu)，包括腺體結(jié)構(gòu)、細(xì)胞結(jié)構(gòu)、亞細(xì)胞結(jié)構(gòu)、血管結(jié)構(gòu)均符合在體內(nèi)的正常狀態(tài)。 此外人乳腺組織在小鼠體內(nèi)能保留正常乳腺組織在相關(guān)激素的刺激下泌乳的功能。免疫組織化學(xué)染色顯示出移植的人乳腺基底膜-肌上皮穩(wěn)定性也發(fā)生了從適應(yīng)到恢復(fù)正常的時(shí)間相關(guān)性變化。Ki67表達(dá)穩(wěn)定，提示人乳腺組織在小鼠體內(nèi)增殖性保持穩(wěn)定。Aromatase及STS染色均呈陰性，提示移植后的人乳腺組織并未在局部合成雌激素，其維持自身結(jié)構(gòu)和功能及促進(jìn)激素受體陽性腫瘤細(xì)胞成瘤的主要因素仍值得深入探究。 小鼠MFP結(jié)構(gòu)特征及腫瘤植入組成瘤失敗提示小鼠MFP及皮下與人體乳腺癌發(fā)生環(huán)境差異較大，不適于乳腺癌生長轉(zhuǎn)移；在小鼠皮下種植的正常人乳腺組織可以存活，不但能長期保持正常的組織結(jié)構(gòu)及功能，還能產(chǎn)生特定的發(fā)育改變，尤其提示乳腺正常微環(huán)境和腫瘤微環(huán)境差別巨大，在選擇模型時(shí)，應(yīng)結(jié)合研究目的充分考慮這一點(diǎn)。
[Abstract]:The incidence of breast cancer is increasing, which seriously threatens the life and health of women. The research on the pathogenesis and prevention of breast cancer is widely carried out. The animal model has an important role in the research of breast cancer. At present, the model of more used mice has its own advantages and disadvantages, the most important defect is that it can not provide human source. Microenvironment, a supportive network composed of various components, is becoming more and more important in the tumor. In this laboratory, the model of human breast human breast and human breast human bone breast cancer in situ and metastasis has been established by SCID (severe combined immunodeficiency) mice, in addition to better simulation of tumor occurrence and transformation. In order to make a further understanding and interpretation of the advantages of a human derived series of models, to make a quantitative analysis of the parameters of the model itself in order to make the model better used, we have adopted a number of technical means for human derived grafts in mice. Changes in structure and function and suitable time for use during adaptation, recovery, normal maintenance and aging were monitored and studied.
In this study, we implanted the human mammary tissue into SCID mice according to the standard operation of the model, then sacrificed mice and removed the normal human breast tissue from the body every week, and performed routine pathological observations of HE staining, transmission electron microscopy and Calponin, P63, Ki67, Aromatase and steroid sulfates (steroid sulfatas). E, STS) immunohistochemical staining. Meanwhile, the mice breast fat pad was compared. In addition, we set up a "tumor implant group" to transplant fresh human breast cancer tissue subcutaneously in SCID mice and observe the changes of tumor tissue subcutaneously in mice after 8 weeks.
First weeks after the operation, the mammary gland of the implanted site of the mice had been encapsulated, and the implanted tissue felt soft. With the growth of the implantation time, the volume of the implanted tissue gradually narrowed and toughened.HE staining. The morphological changes of the mammary gland implanted in the human mammary gland had a tendency: the epithelial cells were swollen and deformed in the first few weeks, and the structure of the glands was blurred. The morphology of glandular and epithelial cells reached the worst after the first 1-3 weeks. From the fourth week of implantation, the morphology of the glands improved, the adipose degeneration in the epithelial cells decreased, the nucleus morphology was regular. At the time of 5 weeks, the gland was close to the shape of the gland before the implantation, and the gland and the epithelial cell structure of the implant were maintained for the next 13 weeks. This normal gland tube structure. The interstitial tissue of the tissue under the light microscope always shows fibrosis and hyaline degeneration, which is not like the time changing trend of the gland structure.
The transmission electron microscope observed the change trend consistent with the ordinary optical microscope, and the structure was first changed and then recovered: the cell nucleus of the epithelial cells was obviously abnormal in 1 weeks, the fat degeneration was obvious in 2 weeks, the fat vacuoles were visible in the cells, the lipid inclusion bodies could be seen in the nucleus, and the organelle in the cytoplasm was not obvious; the cytoplasmic inner line at 3 weeks. The swelling of the mitochondria was obvious, the mitochondria swelled and the mitochondrial crista was clear for 4 weeks. The epithelial cell gap was clearly visible at the beginning of the 5 week, and the sign of the glandular epithelium was connected and the cytoplasm was abundant in the cytoplasm. In the next few weeks, the epithelial cells maintained this kind of structure similar to the normal mammary epithelial cells until the end point. We were specimens for 6 weeks. The cells resembling the expression of the mammary epithelium in the late stage of pregnancy were observed, with abundant organelles in the cytoplasm and a large number of vesicles and endocrine, containing two substances with different electron densities.
In addition to the enveloped vascular network seen in general specimens, red blood cells can be seen in vascular structure.HE slices by light microscopy and electron microscopy, suggesting that these vessels have normal function.
Calponin and P63 are the markers of myoepithelial cells, which can indicate the stability of the local mammary microenvironment. In normal reference, there are complete myoepithelial cells surrounded by the adenoid tube. But in the specimens 1-2 weeks after implantation, the Cal part of the canal is absent, suggesting the absence of the myoepithelial cells, and the nucleus staining index P63 has no obvious changes in.Ki67 before implantation. The expression was scattered in the mammary glands and the implanted mammary glands, and there was no obvious increase in expression and loss of expression. The two routes of local estrogen producing Aromatase and steroid sulfatase, STS were milk before and after transplantation. The staining was negative in all the glands.
All the mice selected in this experiment were unmated mice, so the mammary tissues of the mice were always displayed at the static stage of the mammary gland. The main body of the fat pad was lipid composition, the middle of the duct was mixed with the structure of the ducts, and a small amount of interstitial cells were seen around the catheter, and no acinar structure was found in the section.
In the tumor implantation group, 6 mice were inoculated, 6 cases were all failed, with the increase of the implantation time, the mass was obviously smaller than that of the human breast cancer tissue around and within the.HE staining. In the membrane, the necrotic region appears in the center of the tumor.
The normal human breast tissue has undergone the time related changes in the adaptation period (1-3W) - recovery period (4W) - the four stages of the normal structure maintenance period (5w-13w) - aging period. After the recovery period, the human mammary tissue in the mice can maintain the normal human mammary structure for a long time, including the gland structure and the cell junction. The subcellular structure and vascular structure conform to the normal state of the body.
In addition, human breast tissue can retain the function of normal mammary tissue in the body of the mammary gland under the stimulation of related hormones. Immunohistochemical staining shows that the stability of the basal membrane and myoepithelial cells of the human mammary gland of the transplanted human is also stable from the time of adaptation to the recovery of normal, which suggests that the human breast tissue is in the mouse body. The proliferation retention and stability of.Aromatase and STS staining are negative. It suggests that the human mammary tissue after transplantation is not locally synthesized. The main factors for maintaining its structure and function and promoting the tumor cell formation of the hormone receptor positive tumor cells are still worth exploring.
The structural characteristics of MFP and the failure of tumor implantation tumor suggest that the environment of MFP and human breast cancer in mice is different from the human breast cancer, which is not suitable for the growth and metastasis of breast cancer. The normal human breast tissue in the mice subcutaneous can survive, not only can maintain normal structure and function for a long time, but also produce specific developmental changes, especially in mice. It suggests that there is a great difference between normal microenvironment and tumor microenvironment of breast, which should be fully considered in choosing model.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R737.9;R-332
[Abstract]:The incidence of breast cancer is increasing, which seriously threatens the life and health of women. The research on the pathogenesis and prevention of breast cancer is widely carried out. The animal model has an important role in the research of breast cancer. At present, the model of more used mice has its own advantages and disadvantages, the most important defect is that it can not provide human source. Microenvironment, a supportive network composed of various components, is becoming more and more important in the tumor. In this laboratory, the model of human breast human breast and human breast human bone breast cancer in situ and metastasis has been established by SCID (severe combined immunodeficiency) mice, in addition to better simulation of tumor occurrence and transformation. In order to make a further understanding and interpretation of the advantages of a human derived series of models, to make a quantitative analysis of the parameters of the model itself in order to make the model better used, we have adopted a number of technical means for human derived grafts in mice. Changes in structure and function and suitable time for use during adaptation, recovery, normal maintenance and aging were monitored and studied.
In this study, we implanted the human mammary tissue into SCID mice according to the standard operation of the model, then sacrificed mice and removed the normal human breast tissue from the body every week, and performed routine pathological observations of HE staining, transmission electron microscopy and Calponin, P63, Ki67, Aromatase and steroid sulfates (steroid sulfatas). E, STS) immunohistochemical staining. Meanwhile, the mice breast fat pad was compared. In addition, we set up a "tumor implant group" to transplant fresh human breast cancer tissue subcutaneously in SCID mice and observe the changes of tumor tissue subcutaneously in mice after 8 weeks.
First weeks after the operation, the mammary gland of the implanted site of the mice had been encapsulated, and the implanted tissue felt soft. With the growth of the implantation time, the volume of the implanted tissue gradually narrowed and toughened.HE staining. The morphological changes of the mammary gland implanted in the human mammary gland had a tendency: the epithelial cells were swollen and deformed in the first few weeks, and the structure of the glands was blurred. The morphology of glandular and epithelial cells reached the worst after the first 1-3 weeks. From the fourth week of implantation, the morphology of the glands improved, the adipose degeneration in the epithelial cells decreased, the nucleus morphology was regular. At the time of 5 weeks, the gland was close to the shape of the gland before the implantation, and the gland and the epithelial cell structure of the implant were maintained for the next 13 weeks. This normal gland tube structure. The interstitial tissue of the tissue under the light microscope always shows fibrosis and hyaline degeneration, which is not like the time changing trend of the gland structure.
The transmission electron microscope observed the change trend consistent with the ordinary optical microscope, and the structure was first changed and then recovered: the cell nucleus of the epithelial cells was obviously abnormal in 1 weeks, the fat degeneration was obvious in 2 weeks, the fat vacuoles were visible in the cells, the lipid inclusion bodies could be seen in the nucleus, and the organelle in the cytoplasm was not obvious; the cytoplasmic inner line at 3 weeks. The swelling of the mitochondria was obvious, the mitochondria swelled and the mitochondrial crista was clear for 4 weeks. The epithelial cell gap was clearly visible at the beginning of the 5 week, and the sign of the glandular epithelium was connected and the cytoplasm was abundant in the cytoplasm. In the next few weeks, the epithelial cells maintained this kind of structure similar to the normal mammary epithelial cells until the end point. We were specimens for 6 weeks. The cells resembling the expression of the mammary epithelium in the late stage of pregnancy were observed, with abundant organelles in the cytoplasm and a large number of vesicles and endocrine, containing two substances with different electron densities.
In addition to the enveloped vascular network seen in general specimens, red blood cells can be seen in vascular structure.HE slices by light microscopy and electron microscopy, suggesting that these vessels have normal function.
Calponin and P63 are the markers of myoepithelial cells, which can indicate the stability of the local mammary microenvironment. In normal reference, there are complete myoepithelial cells surrounded by the adenoid tube. But in the specimens 1-2 weeks after implantation, the Cal part of the canal is absent, suggesting the absence of the myoepithelial cells, and the nucleus staining index P63 has no obvious changes in.Ki67 before implantation. The expression was scattered in the mammary glands and the implanted mammary glands, and there was no obvious increase in expression and loss of expression. The two routes of local estrogen producing Aromatase and steroid sulfatase, STS were milk before and after transplantation. The staining was negative in all the glands.
All the mice selected in this experiment were unmated mice, so the mammary tissues of the mice were always displayed at the static stage of the mammary gland. The main body of the fat pad was lipid composition, the middle of the duct was mixed with the structure of the ducts, and a small amount of interstitial cells were seen around the catheter, and no acinar structure was found in the section.
In the tumor implantation group, 6 mice were inoculated, 6 cases were all failed, with the increase of the implantation time, the mass was obviously smaller than that of the human breast cancer tissue around and within the.HE staining. In the membrane, the necrotic region appears in the center of the tumor.
The normal human breast tissue has undergone the time related changes in the adaptation period (1-3W) - recovery period (4W) - the four stages of the normal structure maintenance period (5w-13w) - aging period. After the recovery period, the human mammary tissue in the mice can maintain the normal human mammary structure for a long time, including the gland structure and the cell junction. The subcellular structure and vascular structure conform to the normal state of the body.
In addition, human breast tissue can retain the function of normal mammary tissue in the body of the mammary gland under the stimulation of related hormones. Immunohistochemical staining shows that the stability of the basal membrane and myoepithelial cells of the human mammary gland of the transplanted human is also stable from the time of adaptation to the recovery of normal, which suggests that the human breast tissue is in the mouse body. The proliferation retention and stability of.Aromatase and STS staining are negative. It suggests that the human mammary tissue after transplantation is not locally synthesized. The main factors for maintaining its structure and function and promoting the tumor cell formation of the hormone receptor positive tumor cells are still worth exploring.
The structural characteristics of MFP and the failure of tumor implantation tumor suggest that the environment of MFP and human breast cancer in mice is different from the human breast cancer, which is not suitable for the growth and metastasis of breast cancer. The normal human breast tissue in the mice subcutaneous can survive, not only can maintain normal structure and function for a long time, but also produce specific developmental changes, especially in mice. It suggests that there is a great difference between normal microenvironment and tumor microenvironment of breast, which should be fully considered in choosing model.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R737.9;R-332
【共引文獻(xiàn)】
相關(guān)期刊論文 前3條
1 張軍榮;穆繼英;楊愛軍;尚立娜;牛茜;;5-氟尿嘧啶篩選殘存結(jié)腸癌細(xì)胞的生物學(xué)特點(diǎn)及其與CD133表達(dá)的關(guān)系[J];第三軍醫(yī)大學(xué)學(xué)報(bào);2011年01期
2 夏厚軍;王春艷;張海林;何保麗;角建林;陳策實(shí);;自發(fā)性樹
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