| Part 1: The rat bone marrow mesenchymal stem cells’ isolation, culture, identification and co culture with neonatal rat skin tissueObjective: In vitro rat bone marrow mesenchymal stem cells(BMSCs) were isolated, cultured and amplified to observe the growth morphology, growth curve and the surface markers. The purpose is to cultivate the highly purified BMSCs for co culture with the neonatal rat skin tissue.Methods:1. Compared with several different research methods, we choose the whole bone marrow adherent culture method to isolate, culture and purify the BMSCs. The femoral and tibial bone marrow of healthy SD rats(around 8 weeks, 150g) was drawn out in sterile conditions. After centrifuging and resuspending bone marrow, the cells were inoculated into the culture bottles, which were placed at 37℃, 5%CO2 incubator. High purified, homogeneous cells were obtained by changing medium and cells passage to remove other cells. 2. Through observing and recording the isolated adherent cell growth morphology, we selected the third generation cells in good growing condition to evaluate and record the cells growth curve by MTT method. 3. The expression of cell surface markers(CD34, CD44, CD45, and CD105) was detected by cell immunofluorescence and flow cytometry. 4. Co cultured the neonatal rat skin tissue and passage 3 to passage 5(P3-P5) BMSCs in transwell dish.Results: 1. Observation of the cells growth morphology: Change medium after the cells were inoculated into the culture bottle 24 hours later, then change medium 3-4 days regularly. Observation revealed that the primary adherent cells showed round, spindle, star and other irregular shape. After several passages the cells’ morphology tended to be consistent, which was colony growth in distribution. 2. Determination of cell growth curve: the results showed a typical "S" shape, including the potential adaptation period, logarithmic period and plateau period. 3. Cell surface marker detection: through the two methods we mentioned above, the positive expression rate of CD44, CD105 was extremely high; while CD45, CD34 was very low. Conclusions: 1. Through the whole bone marrow adherent method we successfully isolated and cultured rat BMSCs and determined passage 3 to passage 5(P3-P5) cells’ activity and purity is good. 2. We choose P3-P5 BMSCs of good growth state to co culture with neonatal rats’ skin tissue for the study part 2.Part 2: The establishment of rat full-thickness skin defect model and the healing observation after the implantation of co cultured BMSCs into woundObjectives: 1. Skilled in establishing the rat full-thickness skin defect model. 2. Comparing the wound healing effects between co cultured neonatal rats’ skin tissue with BMSCs and simple BMSCs implantation, we can detect whether co cultured implantation can accelerate the wound healing and explore its mechanism.Methods: 3. The establishment of rat full-thickness skin defect model: 12 healthy SD rats were anesthetized with chloral hydrate by intraperitoneal injection. Each rat back was divided into 3 groups of 2cm diameter rounded full-thickness skin defect wounds on both sides of the spine from head to tail. The wounds were symmetrical distribution and interval was 2cm. 4. Experimental groups: The 6 wounds on each rat back were symmetrical distribution on both sides of the spine. From head to tail in turn it was divided into the skin and BMSCs co culture group(group A), simple BMSCs group(group B) and sterile PBS control group(group C). 5. Cell transplantation: Under the wound center and the edge’s fascia, group A wounds were injected with Brdu labeled BMSCs co cultured with skin tissue, group B were injected with pure Brdu labeled BMSCs, group C were injected with sterile PBS. The number of transplanted cells for A and B group was about 2X107. 6. Wound observation: The wound was observed at 3, 7, 14 and 21 days after injury, including the description of the exudation and proliferation of granulation tissue. The wound healing index calculation(WCI) =(1- the wound area after treatment / original wound area) x100%. 7. The positive expression rate of Brd U, proliferating cell nuclear antigen(PCNA),Integrin beta 1(L-16) in the wound was detected by immunofluorescence method to investigate the wound healing effects.Results: 1. Wound observation: The exudation and proliferation of granulation tissue in wound had occurred in every group. The difference in three groups was not very obvious. But the speed of wound healing between the 3 groups was statistically difference with the SPSS13.0 software by measuring the wound area and calculating wound healing. The wound healing speed from fastest to slowest in turn was Group A, Group B and Group C.2. Histology immunofluorescense results: Brd U labeled BMSCs were observed in both A and B groups and rarely detected in C group. The positive expression of PCNA and integrin beta 1 cells was detected in all three groups. The positive expression of group A and B was more obvious than group C. But the two markers showed the opposite trend. With the increase of time, PCNA positive cells in group A were more than group B, and each group’s positive cells were increasing follow the increasing time. To be opposite, the integrin beta 1 positive expressive cells in group A was less than group B, and the positive cells of each group were reducing follow the increasing days.Conclusion: Compared with simple BMSCs, co culture BMSCs and skin tissue can better improve the wound repair and healing. |
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