| Olfactory ensheathing cells (OECs) are unique glia cells which have similar characteristics with astrocytes and Schwann cells. It can be well integrated into central nervous system and promote regeneration. In addition, it has been found that OECs have other good property such as: neuron protection, remyelination, alleviation astroglia reaction. So OECs were good candidate cell sources for cell transplantation. However, there are still two main problems should be solved: Firstly, non-purified and purified olfactory ensheathing cells were widely used in research. Researchers believe that they chose best purity for their study. Which purity of olfactory ensheathing cells better, purified or non-purified? Still there is no study to compare that in vitro. Secondly, although olfactory ensheathing cells conditioned medium (OECCM) can not protect normal retinal ganglion cells (RGCs) in vitro. Whether OECCM can protect injured RGCs in vitro is still not know. We are design experiments for answering their question.Part I: In order to compare the in vitro survival and growth of OECs of different purities, thus presenting evidence for the optimal purity of OECs for transplantation. OECs of adult SD rats were isolated, cultured, purified and mixed for a purity of approximate 80%, 65%, 50%, 35% with attached fibroblasts. Mixed cells were cultured for another 1 or 3 days. All the cultured cells were stained with anti-p75 immunocytochemistry to verify the initial and subsequent numbers and purities of OECs during cell culture. The morphology of OECs and fibroblasts were observed, the numbers of the total cells and the p75-positive cells were counted, and the purities of OECs were thus obtained. The mean numbers and purities of the p75-positive cells were analyzed statistically. After 1 days culture, the results showed that OECs of different purity show normal morphology. When the culture time increased to 3 days, OECs of 80% demonstrated cell body shrinkage and shorter processes, and the mean number of surviving OECs per observing area decreased remarkably from 35±13 to 23±5 (p=0.02) when the culture time increased from 1 day to 3 days, but no changes were detected in the purity of OECs between 1 and 3 day time points. However OECs of 65% and 50% purity still had plump cell bodies and even longer processes and the mean numbers and purity of surviving OECs presented no dramatically decrease in comparison of those at 1 day. Although OECs of 35% purity showed normal morphology, Mean positive cell number and purity decreased significantly (p<0.05). The value of mean positive cell number was 16±4 and 7±4 respectively at 1 and 3 day. Purity of OECs was 37%±3% and 20%±8% respectively at 1 and 3 day. The results indicated that the survival and growth of cultured OECs of 65%, 50% were predominant to those of other purity, suggesting that purity between 50% and 65% OEC suspension should be considered for an optimal transplantation into the injured central nervous system.PartⅡ: In order to study neuroprotection effects of OECCM on injured RGCs. OECCM obtain from cultured OECs of adult SD rats. After being scratched, experiments can be divided into two parts:⑴Experiments of this part can be divided into two groups (every group n=8): OECCM and control (adding normal medium). After 3 and 6 days culture, we observed RGCs with Phase-contrast microscopy. In order to qualitative and quantitative analysis of RGCs, we stain cultured cells withβ-Ⅲt ubulin.⑵Experiments of this part can be divided into four groups (every group n=8): normal+ OECCM; normal+ control(adding normal medium); scratch+ OECCM; scratch+ control. After 3 and 6 days culture, cell vitality alteration was studied by MTT method. Results:⑴In the group of OECCM, injured RGCs survived and extended their processes to scratch areas. In the control group, we can observe that most body of injured cells were swelling, collapse and degradation, showing necrosis state within scratch areas. Processes retreat back, showing fracture or collapse state. Further, we conduct qualitative and quantitative analysis of RGCs in the injured area. After 3 and 6 days culture, Mean number ofβ-Ⅲtubulin positive cells in the OECCM group(29.67±3.50 and 19.8±2.8)was significantly higher than that in control group(6.81±2.20 and 13±0.6,p<0.05). And 2 mm away from the scratch area, we found positive cells both in the OECCM group or control group have more densely branching processes, there is no obvious scratch impact on cell growth. Statistical shows there is no significantly difference between every group(27.9±2.1,28.6±1.6 and 25.8±6.1,23.8±1.3).⑵MTT results showed that: after being scratched, after adding OECCM 3 days, the OD value is 1.2736±0.0562, significantly higher than that of control group(1.1046±0.1268) (p <0.01). After adding OECCM 6 days, the OD value of cells is 1.1628±0.1599, which higher than that control group (1.1132±0.1128). But there is no significantly difference between OECCM group and control group in normal RGCs(p>0.05).Conclusion: In vitro, OECCM can protect injured RGCs. However, there is no such neuroprotection effect of OECCM on normal RGCs.
|
PDF Full Text Request