| ObjectiveTo find out the culture conditions of the human umbilical cord blood mesenchymal stemcells (UCB-MSCs) ,and the feasibility of healing the brain injured rats by transplanting UCB-MSCs.Methods1.48samples of cord blood were collected.20 of them were from preterm infant,and the others were from term infant.The former was named term of preterm infant,and the latter was named term of term infant.2. Each sample was devided into 2 parts.One part was cultivated by normal culture medium,and the other was cultivated by special culture medium.3.In 48samples, the volume of 5ml was 8,and the volume of 10ml and 20ml was the same 20. Each sample was devided into 2 parts.One part was cultivated by normal culture medium,and the other was cultivated by special culture medium.To find the influence of different sample volume, conceptus age and culture medium to the growth of UCB-MSCs.4. Statistical analysis: The numbers got from differeret culture medium were analysised by SPSS11. 5. The method was crosstabs.There was statistical significance when the value of P was less than 0.05.5.Use male SD rats about 200g to make model.To ligate the right common carotid artery(CCA) and external carotid artery(ECA),and block internal carotid artery(ICA).To inert a line of 0.24mm through CCA and ICA to middle cerebral artery(MCA), and romve it afer 2 hours. The judgement of injury of nerve was from Zea Longa.The content was:0 score:there was no symptom of nerve injury;1 score:the rats could not extend the opposite frontal leg completely;2 score:the rats circled to the negative direction;3 score:the rats tumble to the opposite side;4 core:the rats had no consciousness, and could not walk of them own.6. After 3 days of the model was completed.The MSCs marked by DAPI were transplanted into the brain of the experimental group(EG).And the control group(CG) were transplanted by physiological saline of the same volume.The dose was 5μ1 of UCB- MSCs.And the place was the contralateral lateral ventricle.7. To contrast the neurologic defect scores of the models between EG and CG before transplantation.Repeat the comparison after 28 days of transplantation.8. The behavior change were record and compared between EG and CG.(1)water maze experiment:The experimentation was completed in a drum of 1m diameter,and the water in it was 62cm high.The drum was separated into 4 parts.There was a 60cm high platform of 6cm diameter in the first part of the drum.The rats were trained from the first part to the fourth part 4 times everyday. The time of reaching at the platform was recorded.When the rat was failed in 60s,it would be placed at the platform for 10s.The training was repeated every 30-60s,and would continue for 6 days.In the seventh day,the platform would be renmove to the other part of the drum.The time of the rats to the platform from the first part and the fourth part was recorded.And the time of the rat stopping in the first part was recorded,too. (2) Open box experiment:There was a box of 36cm high,36cm width and 36cm length without a cover.There were 9 checks in the floor divided by black line.The rat was palaced in the middle of the box,and the movement was recorded in 30s.9.Statistical analysis: All the numbers were expressed by (x|-)±S, and were analysised by SPSS11.5.The results of water maze experiment and open box experiment were analysised by independent-samples T test. There was statistical significance when the value of P was less than 0.05. The neurologic defect scores of 1th and 28th were analysised by independent-samples test. There was statistical significance when the value of P was less than 0.05.10.Immunohistochemistry: After the the study of ethology,some brains of the rats were detected the fluorescence of the DAPI,others were added to another fluorescein. After the fixation and section of the brain tissue,some antibody of MAP-2 was added to it.The samples were placed to the box of some humidity and temperature of 37°C about 30min.Then they were washed by 0.01mol/l PBS of Ph7.2 twice.The epactal liquid was removed,and then added IgG antibody marked by FITC.Repeated the steps above ,then we could took photos.Results1.The ralationship between volumes and culture: The volumes of cord blood were 5ml in 8 samples, 10ml in 20 samples and 20ml in the other 20 ones. Each sample was devided into two pairs and cultivated in normal or special culture medium respectively. The anticipant cells were absent in 16 samples with 2.5ml. The adhere cells with round cells or spindle cells were observed in 80 samples with 5 or 10ml volume. In the group of 5ml samples,there were 2 samples were cultivated successfully in normal culture medium.And there were 5 samples were successfully in special culture medium. In the group of 10ml samples,there were 5 samples were successfully in normal culture medium.And there were 7 samples were successfully in special culture medium.The result of statistics was that the values of P were all larger than 0.05 in either normal or special culture. (P=0.407normal,P=0.731special)That meaned there was no fifference in normal or special culture.But the size of 2.5ml were too small to the cells to grow.2. The ralationship between gestation age:There were 20 preterm infants and 28 term infants. 29 failure samples with no adhere cells were observed in both groups. There were 7 samples were cultivated successfully in normal culture medium.And the number of preterm infants within it was 6.There were 17 samples were successfully in special culture medium. And the number of preterm infants within it was 12. The cells were classic fusiform shape which looks like the MSCs. They were small at first, then accreted slowly, at last became heterogeneitic adhere cells with diversific forms. The result of statistics was that the values of P were all smaller than 0.05 in either normal or special culture(P=0.016normal,P=0.001special).That meaned thegestational age was a factor to the grow of UCB-MSCs.3. The ralationship between medium and culture: Among all the 48 samples, there was 5 samples which were all got MSCs no matter in normal medium or special medium.There was 17 samples which got MSCs only in special medium.And there was 7 samples which got MSCs only in normal medium. The result of statistics was that the value of P was larger than 0.05 (P=0.08).That meaned there was no difference in medium..4. The determintation of the CDs on the MSC: CD34,CD45 on haematogenesis cells and CD106 on endotheliocyte were absent on the MSC. The CD105 and CD29 were expressed highly which were similar as the BSC.5. The behavior detection:the rats were part into EG and CG.The number of former was 20, and the number of latter was 18.Two rats in the EG were dead duiring the transplantation,and one in the CG was dead.(1)The result of water maze experiment:The average time of the rats in the EG reaching at the platform was 22.33s,and the average time of the rats in the CG was 30.18s.There was significant(P<0.01, P=0. 000).There were 2 rats in the EG could not reach the platform once.(2)The result of open box experiment:The average time of EG was 1.06s,and the average time of CG was 1.35s.There was no difference (P=0. 354).6.The score of neurologic defect:One day after the model finished,the average score of EG was 3.00,and the average score of CG was 3.18.There was no differerce (P=0. 256) .After 28 days,the average score of EG was 0.75,and the average score of CG was 1.47. There was significant(P<0.05,P=0. 002).7.Histology detection:When the MCA was stopped,the lateral caudate nucleus and the same side cortex were the worst.In some cases, the consecutive cortex might even disappear. The extent and locationb of brain injured in both EG and CG were the same in appearance.The rats which were transplanted by MSCs could be found cells marked by DAPI in the place of transplanting.The cells were found in the place that between the health and injured brain.And the cells distributed dispersed without evidently accumulative feature.Two transplanted rats could not found the marked cells,because the place that ransplantated by MSCs was destroyed.The MSCs could not exist in the necrosis zone.In the brain of the rats of CG,the marked cells could not be founed.ConclusionIn the growth of UCB-MSCs,the the sample size, gestational age and medium were all important.In either normal or special medium,there was no difference in the sample size of 5ml or 10ml.That meaned the sample size from 5ml to 10ml were all right for UCB-MSCs. Gestational age was an important factor in the growth of UCB-MSCs.Though there was no difference in statistics between normal and special medium,we could see that during the growth of the cells,it in the special medium was growing much faster,and the type of cells was simpler.It was feasible of using MSCs to heal the brain injured rats.The UCB-MSCs marked by DAPI before transplantation could also be detected.That meaned the transplanting cells could survive in the new place.Through detecting the fluorescence of DAPI and FITC,It could say that the UCB-MSCs was becoming nerve cells in the brain tissue to play an important role in the treatment.It was certificated by contrasting the animal behavior, brain tissue staining between the rats of EG and CG.There was some therapeutic efficacy by using MSCs to heal brain injured rats. |
PDF Full Text Request