人源性乳腺微环境小鼠模型的移植物功能研究

发布时间：2018-07-31 07:57

【摘要】：乳腺癌发病率日益增长，严重威胁女性生命健康。对于乳腺癌发病机制及防治的研究广泛开展，动物模型这一体内研究工具在乳腺癌的研究中有着重要的作用。目前应用较多的小鼠模型有着各自的优缺点，最主要的缺陷是不能提供人源性微环境。微环境是一个由多种成分构成的支持性网络，其在肿瘤中的重要性日益得到重视。本实验室利用SCID（严重联合免疫缺陷）小鼠先后建立了人乳腺-人乳腺及人乳腺-人骨乳腺癌原位及转移模型，除了能更好的模拟肿瘤发生及转移的各个过程之外，均提供了人源性组织特异性的微环境。为了对人源性系列模型的优势做进一步的理解和阐释，更为了对模型本身各项参数进行定量，以便于该模型更好的被运用，我们采用多种技术手段，对人源性移植物在小鼠体内从适应、恢复、正常维持至衰老的过程中，其结构和功能的变化和适宜使用时间做了监测和研究。本研究中，我们按照构建模型的标准操作将人乳腺组织植入SCID小鼠，之后每周牺牲小鼠并取出体内移植的正常人乳腺组织，分别进行HE染色常规病理观察，透射电镜观测及Calponin、P63、Ki67、芳香化酶（Aromatase）和类固醇硫酸酯酶（steroid sulfatase, STS）的免疫组织化学染色。同时留取小鼠乳腺脂肪垫进行了对比。另外，我们设置了“肿瘤植入组”，将新鲜人乳腺癌组织移植在SCID小鼠皮下，观测植入8周后肿瘤组织在小鼠皮下的改变。术后第1周小鼠植入部位的乳腺已被形成的包膜包裹，植入的组织触感柔软，随植入时间的增长，植入组织体积逐渐缩小变韧。HE染色下人乳腺植入小鼠体内后形态变化具有一定趋势：植入最初几周上皮细胞肿胀变形，腺体结构模糊，这种变化在第1-3周逐渐加重，腺体及上皮细胞形态在第2-3周时达到最差，从植入第4周开始，腺体形态出现好转，上皮细胞内脂肪变性减少，细胞核形态规则，5周时腺体与植入前的腺体形态接近，此后13周，植入的腺体及上皮细胞结构保持基本正常的腺管结构。光镜下植入后组织间质始终呈现纤维化和玻璃样变性，这种变性并不像腺体结构具有随时间的变化趋势。透射电子显微镜观察到与普通光学显微镜相符的变化趋势，结构先变差再恢复：1周的上皮细胞细胞核形态明显异常；2周细胞脂肪变性明显，，细胞内可见大量脂肪空泡，核内也可见到脂质包涵体，胞质内细胞器不明显；3周时胞质内线粒体肿胀明显；4周线粒体肿胀消退，线粒体嵴清晰；5周开始上皮细胞间隙清晰可见，且能观测到腺上皮标志性的桥粒连接，胞质内细胞器丰富，此后数周上皮细胞维持此种类似正常乳腺上皮细胞的结构直至观测终点。我们在6周的标本中观测到了类似妊娠后期乳腺上皮表现的细胞，胞质内细胞器丰富，并且有大量小泡且向腔内分泌，内含两种不同电子密度的物质。除大体标本见到的包膜血管网之外，光镜及电镜均可见到血管结构。HE切片中血管内可见红细胞，提示这些血管具有正常功能。 Calponin及P63是肌上皮细胞的标志物，可提示局部乳腺微环境稳定性。正常参照中腺管周围有完整的肌上皮细胞包围。但植入后1-2周的标本中，腺管周围Cal部分表达缺失，提示肌上皮细胞出现缺失，胞核染色指标P63无明显变化。Ki67在植入前和植入后的乳腺腺体中均呈散在表达，并未随植入时间的推移出现明显的表达增高和表达缺失的情况。局部产生雌激素的两条途径的限速酶芳香化酶（Aromatase）和类固醇硫酸酯酶（steroid sulfatase, STS）抗体在移植前及移植后各时间点的乳腺组织中均呈现染色阴性。本实验选用的均是未经交配的小鼠，故小鼠的乳腺组织始终处呈现静止期乳腺的表现，脂肪垫内主体为脂质成分，中间夹杂导管结构，导管周围可见少量间质细胞，切片中未见腺泡结构。随实验进行，小鼠乳腺结构未出现时间相关性改变。肿瘤植入组共接种6只小鼠，6例成瘤均失败，随植入时间的增长肿块均明显缩小，手术取出的肿块体积明显小于植入前。HE染色下移植后的人乳腺癌组织周围及内部可见血管，血管内可见到红细胞。肿瘤组织周围有包膜，肿瘤生长局限在包膜内，部分肿瘤中心出现坏死区域。正常人乳腺组织在移植到小鼠体内后经历了适应期（1-3w）—恢复期（4w）—正常结构维持期（5w-13w）—衰老期四个阶段的时间相关性变化。恢复期后，小鼠体内的人乳腺组织可在相当长的时间内维持人类正常乳腺结构，包括腺体结构、细胞结构、亚细胞结构、血管结构均符合在体内的正常状态。此外人乳腺组织在小鼠体内能保留正常乳腺组织在相关激素的刺激下泌乳的功能。免疫组织化学染色显示出移植的人乳腺基底膜-肌上皮稳定性也发生了从适应到恢复正常的时间相关性变化。Ki67表达稳定，提示人乳腺组织在小鼠体内增殖性保持稳定。Aromatase及STS染色均呈阴性，提示移植后的人乳腺组织并未在局部合成雌激素，其维持自身结构和功能及促进激素受体阳性肿瘤细胞成瘤的主要因素仍值得深入探究。小鼠MFP结构特征及肿瘤植入组成瘤失败提示小鼠MFP及皮下与人体乳腺癌发生环境差异较大，不适于乳腺癌生长转移；在小鼠皮下种植的正常人乳腺组织可以存活，不但能长期保持正常的组织结构及功能，还能产生特定的发育改变，尤其提示乳腺正常微环境和肿瘤微环境差别巨大，在选择模型时，应结合研究目的充分考虑这一点。
[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.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R737.9;R-332

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