摘要
目的
结膜是眼表组织的重要组成部分，对维持眼表的完整和泪膜的稳定至关重要。当因外伤或自身疾病导致结膜细胞丢失和功能丧失时可使泪液膜的完整性破坏进而引起瘢痕形成、睑球粘连和眼球运动受限。这是一类难治性的严重致盲性眼表疾病。目前常规的治疗方法是用正常的结膜或结膜替代物行结膜重建。目前应用于临床的结膜重建包括自体结膜移植、唇粘膜移植、异体结膜移植、羊膜移植等，但各种方法都有不同的缺点。最理想的方法是利用组织工程方法，选择适当的种子细胞，培养构建具有正常生理功能的结膜组织片。
人骨髓间充质干细胞(human mesenchymal stem cells，hMSCs)是骨髓中与造血干细胞(hematopoietic stem cells，HSCs)不同的，具有向骨组织、软骨组织、脂肪组织、肌肉组织等多种组织细胞分化的成体干细胞。hMSCs在体外可以大量增殖，并具有克隆形成的能力。由于其具有多向分化的潜能，在体外又较容易培养扩增，加之具有潜在的细胞治疗价值，故成为生物学界和医学界的研究热点。本研究分离培养和鉴定人的结膜上皮细胞、hMSCs，利用不同的诱导条件对hMSCs进行诱导分化，为临床治疗结膜疾病提供一个新思路。
方法
一、正常人结膜细胞的培养与鉴定
1、材料和方法
结膜取自中国医科大学附属一院眼科角膜取材后的供体眼球。供体均为身体健康，无眼病史的正常人。平均年龄34.5岁。死后4h内取材。采用消化后组织块法培养结膜原代细胞，并进行传代。
2、培养结膜细胞的免疫组化鉴定
把第2代的结膜细胞接种于置在培养皿中的盖玻片上，当细胞接近融合时，及时取出作原位甲醇固定，CK13单抗、DAB显色免疫组化染色。
3、结膜细胞生长曲线的测定
随机选取第1、3、5传代细胞，以5×10~4／ml密度传代、分别培养1-9天，每天分别细胞计数并进行分析比较，重复3次。结果以均数土标准差((?)+s)表示。
4、培养结膜细胞的RT-PCR检测
总RNA抽提试剂盒(上海生工生物工程技术服务有限公司)提取培养结膜上皮细胞总RNA，-70℃保存备用。按照NCBI公布的Muc4、Muc5ac基因序列，设计引物。分别加入不同引物，PCR终体系25μl。取PCR产物10μl经2％的琼脂糖凝胶电泳。
二、人骨髓间充质干细胞的培养及鉴定
1、材料和方法
骨髓来源：来自临床检查骨髓的正常结果者，平均年龄40.6岁。采用全骨髓贴壁法培养骨髓间充质干细胞，并进行传代观察。
2、培养骨髓间充质干细胞的免疫组化鉴定
把第3代的骨髓间充质干细胞接种于置在培养皿中的盖玻片上，当细胞接近融合时，用CD44单抗、DAB显色免疫组化染色。
3、培养骨髓间充质干细胞生长曲线的测定
随机选取第1、4、8传代细胞，以1×10~4／ml密度传代，分别培养1-9天，每天分别细胞计数并进行分析比较，实验重复3次。结果以均数±标准差((?)±s)表示。
4、流式细胞仪检测
取扩增传代的1，3，5代细胞。CD34-PE，CD45-FITC，CD44-FITC，CD29-PE单克隆抗体，使用同型对照单克隆抗体确定背景标记，流式细胞仪分析5000个细胞，检测阳性率。重复3次，结果用均数士标准差((?)±s)表示。
三、骨髓间充质干细胞向结膜样细胞诱导分化的研究
1、人结膜上皮细胞和骨髓间充质干细胞的共同培养
将传代后的P2-P4代骨髓间充质干细胞按4～5×10~4／ml的细胞密度接种于六孔Transwell培养板底层(预置盖玻片)，将传代后的P2-P3代结膜上皮细胞按6×10~4／ml接种于Transwell培养板上层，共同培养11天。
2、共同培养骨髓间充质干细胞免疫组化鉴定
共同培养11天后，底层培养细胞达到70-80％左右融合时，取出Transwell培养板底层的盖玻片，CK13单抗、DAB显色免疫组化染色。
3、共同培养骨髓间充质干细胞RT-PCR检测
提取共同培养11天骨髓间充质干细胞总RNA，-70℃保存备用。按照NCBI公布的Muc4、Muc5ac基因序列，设计引物。分别加入不同引物，PCR终体系25μl。取PCR产物10μl经2％的琼脂糖凝胶电泳。
结果
一、正常人结膜细胞的培养与鉴定
1、活体细胞的形态
消化后组织块培养法：48小时后见大量上皮细胞贴壁，细胞不规则呈圆形、椭圆形、多边形，细胞体肥大透亮，细胞核位于中央，胞膜清楚，约12-14天后细胞呈镶嵌状排列，融合成膜状。
2、免疫组化鉴定
培养的人结膜上皮细胞CK13免疫组化染色见阳性颗粒均匀分布于胞浆中。
3、培养结膜细胞生长曲线
传代培养多数样本于P5代以后开始出现衰老征象。传代细胞的生长较原代要慢些，传代培养潜伏期约为48～60h；传代培养对数增殖期约为3～7d；对数增殖期结束后，结膜细胞生长逐渐缓慢，进入平台期。
4、RT-PCR检测
人结膜细胞Muc4 mRNA、Muc5ac mRNA在培养的正常人结膜细胞中均有表达。
二、人骨髓间充质干细胞的培养及鉴定
1、活体细胞的形态
原代细胞培养72小时后，细胞贴壁，呈纺锤型、梭型、多角形，增值迅速，10天细胞呈长梭形，克隆样生长，达80-90％融合。传代培养细胞形态与原代细胞基本一致，增殖迅速，约7-9天达到80-90％融合。
2、培养骨髓间充质干细胞的免疫组化鉴定
免疫组化CD44染色，骨髓间充质干细胞胞浆阳性染色。
3、培养骨髓间充质干细胞生长曲线的测定
传代培养多数样本于P8代以后开始出现衰老征象。传代细胞的生长较原代要慢些，多于接种后9天左右即可铺满整个培养孔的底面。传代培养潜伏期约为24～36h；传代培养对数增殖期约为4～6d；对数增殖期结束后，细胞生长逐渐缓慢，进入平台期。
4、流式细胞仪检测
流式细胞检测结果表明，所培养的骨髓间充质干细胞表面有粘附分子的表达，CD29，CD44阳性标记出现单峰，造血细胞分化抗原CD34，CD45表达阴性，经扩增传代后的各代骨髓骨髓间充质干细胞表面标记表达阳性率无明显差异(P＞0.05)。
三、骨髓间充质干细胞向结膜样细胞诱导分化的研究
1、人结膜上皮细胞和骨髓间充质干细胞的共同培养
共同培养72小时后，贴壁骨髓间充质干细胞部分呈椭圆形、圆形改变，部分呈梭形。共同培养11天后，大部分细胞呈多边形、圆形、椭圆形，少量细胞呈梭形。
2、共同培养骨髓间充质干细胞免疫组化鉴定
共同培养11天后，CK13单抗，DAB显色免疫组化染色，骨髓间充质干细胞分化细胞CK13免疫组化染色阳性。
3、共同培养骨髓间充质干细胞RT-PCR检测
骨髓间充质干细胞分化细胞Muc4MRNA表达阳性，Muc5acMRNA表达阴性。
结论
1、消化后组织块培养法可以获得正常结膜上皮细胞，2-3代结膜上皮细胞适合其他研究。
2、全骨髓贴壁法培养骨髓间充质干细胞可以获得高纯度的具有正常功能的间充质干细胞，用于诱导分化研究不宜超过4-5代。
3、骨髓间充质干细胞可以诱导分化成结膜样上皮细胞。
4、分化细胞具有部分正常结膜上皮细胞功能。
Objective
Conjunctiva is an important part of ocular surface.It maintains ocular surface integrity and tear-film stabilization. When large areas damaged, the loss of original function can lead to scar formation and symblepharon, eye movement is restricted, it also resulted in the tear film integrity destruction which isone of 'the high incidence of refractory eye diseases causeing of blindness in our country. The effective treatment is conjunctival reconstructs with normal conjunctiva or conjunctival substitute. Conjunctival reconstruction methods including autoallergic or allogenic conjunctival transplantation、amniotic membrane transplantation and lip mucosa transplantation. But all have different shortcomings. By now the most ideal method is to reconstruct the conjunctiva using tissue-engineering methods to get the normal physiological function. Human mesenchymal stem cells(hMSCs) is adult stem cells that can differentiate into the bone tissue, the cartilage organization, the fatty tissue, the muscular tissue and so on the many kinds of organization cell. HMSCs may massively multiply in vitro and has the ability to create clones. Because it has multidifferentiation potential and easily cultured in vitro, hMSCs has potential therapeutic value and has become biology and medical research hot spot, In this study, hMSCs has been differentiated into normal conjunctival epithelial cells, providing a new idea for the clinical treatment of conjunctival diseases.
Methods
一. Normal conjunctival cells culture and Identification
Material and method
Conjunctiva obtained from China Medical University Hospital eye cornea transplant donor's eyeballs. The donors are all healthy persons without eye medical history. The average age of donor was 34.5 years. Enucleated within 24 hours after death.
2. immunohistochemical identification of cultured conjunctival cells
When the second cultured generation of conjunctival cells close to confluence, the glass coverslips were removed in situ fixation by methanol and DAB color immunohistochemical staining by CK13 monoclonal antibody in time.
3. growth curve of conjunctival cells
The 1st,3rd,5th passage cells were selectecd randomly and subcultured at the density of 5×10~4/ml,after culturing for another 1-9 days, were counted and compared the cell number every day. Each experiment was repeated 3 times and data were shown in mean±standard deviation.
4. RT-PCR test of conjunctival cells
Total RNA of cultured conjunctival cells was extracted by total RNA extract kit(sangon biological engineering technology,shanghai) and stored at-70℃. Primer of Muc4、Muc5ac were designed according to the gene order publicated by NCBI. 25μl PCR system with different primer was carried out. 10μl of each PCR products were examined by 2% agarose gel electrophoresis.
二. Culturing and assessment of human bone mesenchymal stem cells
1. Material and method
Bone marrow were derived from the normal persons in clinical myeloid examination and the average age was 40.6. Bone mesenchymal stem cells were cultured and subpassaged by the means of total bone marrow adherence.
2. Immunochemistry identification of cultured Bone mesenchymal stem cells
The 3rd passage of bone mesenchymal stem cells were seeded on cover glass soaked in culture capsule. Monoclone antibody against CD44 was used to identify the near confluent cells and the positive stain was shown by DAB.
3. growth curve of bone mesenchymal stem cells
We selectecd the lst,4th,8th passage cells randomly and subcultured at the density of 1×10~4/ml, Continue to culture them for 1-9 days, then count and compare the cell number every day. Each experiment was repeated 3 times and data were shown in mean±standard deviation.
4. Flow cytometry test
The amplified and subcultured bone mesenchymal stem cells of passage 1,3,5 were identified by monoclone antibody against CD34-PE, CD45-FITC, CD44-FITC,CD29-PE. Positive rate of 5 thousands mesenchymal stem cells were analysed by flow cytometry test. Each experiment was repeated 3 times and data were shown in mean±standard deviation.
三. Study on the differentiation of bone mesenchymal stem cells to conjunctiva cells
1. Coculture of human bone mesenchymal stem cells and conjunctiva cells
Subcultured mesenchymal stem cells of passage 2-4th were seeded on the bottom of transwell culture plate at the density of 4～5×10~4/ml. Subcultured conjunctiva cells of passage 2-3rd were seeded on the upper tier of transwell culture plate at the density of 6×10~4/ml and cocultured for 11 days.
2. Immunochemistry examination of cocultured bone mesenchymal stem cells
After cocultured for 11 days, nearly 70-80% confluent bone mesenchymal stem cells were tested by monoclone antibody against CK13.The positive stain was shown by DAB.
3. RT-PCR test of cocultured bone mesenchymal stem cells
Total RNA of cocultured bone mesenchymal stem cells were extracted and stord at-70℃. Primer of Muc4、Muc5ac were designed according to the gene order publicated by NCBI. 25μl PCR system with different primer was carried out. The result was checked by 2% agarose gel electrophoresis with 10μl PCR products.
Results
一. Culturing and identification of human conjunctiva cells
1. morphous of conjunctiva cells
Cells were harvested by tissue cultivation after digestion. Ponderosus cells adhered after 48 hours. Cells were shown in shape of round, ellipse and polygon. Cell body was loose and lucency with nucleus in center, cell membrane was also clearly seen. Cells were arranged in inlay and fuse in film after 12-14 days.
2. Immunochemistry test
Nomogeneous immunochernical positive granules against CK13 were observed in endochylema of human conjunctiva cells.
3. growth curve of conjunctival cells
Subcultured cells were aged and feeble after 5th passage. Subcultured cells grew slowlier than primary cells and the latent phase for subcultured was 48～60h. Logarithm proliferation phase for subcultured cells was 3～7d. Cells entered flat form and grew slowly after logarithm proliferation phase.
3. RT-PCR test
mRNA of Muc4, Muc5ac were all transcripted in human conjunctiva cells.
二. culturing and tabling of Bone MSCs
1. morphous of bone mesenchymalstem cells
primary MSCs adhered after cultured for 72 hours, the cultured cells were characterized by spindle-shaped, shuttle,polygon appearance and proliferated rapidly. Fusiform cells reached 80-90% confluent after 10 days and grown in clone. Subcultured cells nearly took the same shape of primary cells, and proliferated rapidly. Cells reached 80-90% confluent after 7-9 days
2. Immunochemistry identification of cultured Bone mesenchymal stem cells
MSCs cytoplasm were stained positively by CD44 monoclonal antibody.
3. growth curve of bone mesenchymal stem cells
Subcultured cells were aged and feeble after 8th passage. Subcultured cells grew slowlier than primary cells and reached confluence after 9 days.The latent phase for subcultured was 24～36h. Logarithm proliferation phase for subcultured cells was 4～6d. Cells entered flat form and grew slowly after logarithm proliferation phase.
4. flow cytometry test
Data were shown that cultured bone mesenchymal stem cells had adhere molecular expression. Unimodality was shown in CD29, CD44, and negative results were shown in hematopoietic cell differentiation antigen-CD34, CD45. There was no significient difference in positive rate between subcultured MSCs (P＞0.05).
三. Study on the differentiation of bone mesenchymal stem cells to conjunctiva cells
1. Coculture of human bone mesenchymal stem cells and conjunctiva cells
Part MSCs were shown in ellipse,round and part were fusiform after cocultured for 72 hours. Cells were polygon and round ellipse in bulk, and only a small part were fusiform.
2. Immunochemistry study of cocultured bone mesenchymal stem cells
After cocultured for 11 days, nearly 70-80% confluent bone mesenchymal stem cells were tested by monoclone antibody against CK13 and the positive stain was shown by DAB. Positive stain against CK13 was shown in differentiated bone MSCs.
3. RT-PCR test of cocultured bone mesenchymal stem cells
Muc4 mRNA was transcripted by Differentiated bone MSCs, but Muc5ac was not.
conclusion
1. Conjunctiva cells can obtain by tissue cultivation after digestion and the 2-3rd passage subcultured cells were suitable for research.
2. Highly purified normal mesenchumal stem cells can obtain by means of total bone marrow adherence and the most suitable passage for differentiation was within 4-5 passage.
3. Bone mesenchymal stem cells can be differentiated into conjunctiva cells.
4. Differentiated MSCs cells have partial function of normal conjunctiva cells.
引文
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