| There are three parts in this study about the biological characters of the bone marrow -derived neural stem cells(BMSCs-D-NSCs). First, we culture the BMSCs-D-NSCs and the embryonic brain-derived neural stem cells(EB-D-NSCs) by the special neural stem cell culture medium, and study their cell biologic characters respectively. Second, we observe the two kinds of cells' shape in different stage by the atomic force microscopy(AFM), compare their surface appearance .Third , we study the influence and mechanism towards the proliferation and differentiation of the BMSCs-D-NSCs by influencing the expression of JAK/ STAT signal pathway.Chapter I:Research on the biological characters and culture of the rat neural stem cells in vitro Part I:Research on the biological characters and culture of the BMSCs-D-NSCs in rat Objective: To establish the culture system in vitro of inducing and purifying the BMSCs-D-NSCs .observe the biological characters of the BMSCs-D-NSCs. Methods: 1, Culture and purify the bone marrow stromal cells: separation the bone marrowstromal cells by density centrifugation , adding bFGF20ng/ml RA300ng/ml in the special bone marrow neural stem cell culture medium, culture for 6 days, culture and purify the cells after beating the cells sticking to the wall.2, Detect the proliferation of the cells applying the MTT method,.3, Detect the proliferation cycle of the cells by the flow cytometer.4, Detect the surface marker CD29. CD34 CD44 CD45 by the flow cytometer.5, Marking the culture cells with nestin, induce the cells to different iate to be neuron and glial cells by the special bone marrow neural stem cell culture medium ( contain 10% calf serum, BDNF20ng/ ml), examining the expression of NSE, GFAP respectively applying immunocytochemistry method.Results:1, The more purified bone marrow neural stem cells can be obtained by density centrifugation method and sticking wall method ,for the effection of the special bone marrow neural stem cell culture medium and the inducer - bFGF> RA.2, The expression of nestin the marker of the nerve stem cells can be detected in the bone marrow neural stem cell;And the cells can be induced to be neuron and neuroglial cells by BDNF-. serum, which expresse NSE and GFAP.3, Detecting by flow cytometer ,we can find 90.51% of the bone marrow neural stem cells express the surface marker-CD29 or CD44 and 92.88% do not express CD34, CD45,which hints that the bone marrow neural stem cells is pure and the bone marrow neural stem cells come from the bone marrow stromal cells mainly not the hematopoietic cells .4, The growth curve and results of the detection of cell's cycle shows that the subculturing bone marrow neural stem cells proliferation slowly , the peak is in the 6th day; the cells mainly stay in the period of Gl/ GO, the cell's proliferation exponentponent is 17.5%.Part IIResearch on the biological characters and culture of the EB-D-NSCs in rat Objective: To establish the culture system in vitro of inducing the EB-D-NSCs by the special neural stem cell's culturing medium, and bserve the biological characters of the EB-D-NSCs. Methods:1, primary culture of the embryonic stem cells of rat: take out the embryo of the 10.5-14.5 days gestational aged SD rat sterility, separating the constitution of the prosencephalon cortex to make the unicellular liquid, culture in the specially nerve cell medium( contain the bFGF20ng/ml without serum), collect the subcultured cells by centrifugation 7 days later.2, observe the growth of the primary and subcultured cells through the phase contrast microscope.3, Count the cell and draw growth curve: Draw the growth curve after continuously counting the cells for 8 days.4, Detect the subcultured cells' cycle by flow cytometer.5, Induce the embryonic stem cells to differentiate: add the serum of embryonic cattle ,make the last density be 10%.6, The cellular immunohistochemistry: Stain the NSCs with nestin> GFAP or NSE according the normal immunocytochemistry method after primary culture > subculture and differentiate.Results:1, The neural stem cell of the prosencephalon cortex can be separated successfully by the special neural stem cell culture medium. The neural stem cell of primary culturing can stay in the undifferentiated station under the effection of the bFGF ,the cells show big round and pinch form in the serum-free medium andgather like a ball;and the nerve stem cells gather in the ball;some of the big round cells move out the ball and some differentiate;there are neuron and neuroglial cells grow around the ball;the special antigen of the big round cells in the ball can be stained positively with nestin .2, the neural stem cell in the ball is still big and round after centrifu gation and subculture, positively stained by nestin.Comparing with the bone marrow neural stem cell,these cells can gather more easily and form clone mass. These cells express the special antigen- NSE and GFAP after induced by the serum-free medium ,which shows the embryonic stem cell could multi -differentiate.3, the growth curve shows the nerve stem cell's number raise continuously after primary culture and subculture ,the peak of proliferation on the fifth day .it shows that the NSCs cultured in vitro can be segmentation continuously and can self-renewing and breeding . The cells mainly stay in the cycle of GO/ Gl, but the proliferation exponent 32% is higher than the BMSCs-D-NSCs.Conclusions of chapter one:1, The BMSCs-D-NSCs can be induced to form by using the special neural stem cell culture medium and purified by density centrifugation and subculturing. The cells come mainly from the bone marrow stromal cell. The biological characters show it stays in the cell cycle of Gl/ GO, the cell's proliferation index is 17.5% ,the cell can proliferate and can be induced into the nerve like cell.2, The EB-D-NSCs can be induced to form by using the special neural stem cell culture medium .The cells gather in the nests matter like a ball and form clone during primary culture ,the cell's growth curve and detection of the cell cycle shows stronger ability to proliferate than the BMSCs-D-NSCs.3, Both the BMSCs-D-NSCs and the EB-D-NSCs appear big round cells whenculture by the special neural stem cell's cuture medium, they have the similar form and can be positively stained by the special marker of the neural stem cell, both the cell can be induced to be neuron and neuroglial cells, which expresse the special antigen of nerve stem cell- NSE and GFAP;It shows that the two kind of nerve stem cells have the ability to multidifferentiate.Chapter IIObservation of BMSCs-D-NSCs and EB-D-NSCs in rat under AFMObjective:To explore the method of observing cell by AFM, and observe the form of differentorigin neural stem cell(bone marrow and the embryonic brain ) in different stage ofthe culture and differentiation, and compare the two kind of neural stem cell.Methods:1,Culture and induce the two kind of neural stem cells respectively.2,Detect the ability of forming the cell clone by the two kind of neural stem cells .3, Observe the neural stem cell of different stage during culture and differentiation by the AFM.4, Detect the difference of the grain degree of the two kinds of cells' surface by the self-analysis software of the AFM.Results:1, the EB-D-NSCs shows stronger ability to form clone than the BMSCs-D-NSCs , which mean it can proliferate more strongly.2, Observe the different stage of the BMSCs-D-NSCs and differentiate into neural cell like by AFM:The BMSCs-D-NSCs is 7.5-12.8 urn (10.3 ±1.2 urn) and round, it form the neural cell like with big and short axon, the neurite of the neuroglial cell is thic and big .3, Observe the cultured EB-D-NSCs and the differentiated neural cells by AFM, the neural stem cells' diameter is 7.0- 11.8um(9.2±1.8 um), big and round , the shape is similar to the BMSCs-D-NSCs, but the neuron and neuroglial cell origined from EB-D-NSCs are more mature than those origined from the BMSCs-D-NSCs4, The average rough degree and average square rough degree of the cell's surface under the AFM can be basic statistics parameter of the cell's superficial rough degree.5, It shows the cell surface grain of the BMSCs-D-NSCs is larger and the surface is rougher than the one of EB-D-NSCs detected by the AFM , which may have something to do with its having stronger differentiating ability.Conclusions of chapter II:1, The AFM is a kind of more convenient and direct tool to observe cell .It providesclearer and more detailed image of the cell, it helps to observe cells from variousaspect with itself software.2,The BMSCs-D-NSCs and EB-D-NSCs have similar appearance under theAFM ,but the neural cell like differentiated from the EB-D-NSCs is more mature andmore exquisite than the one from the BMSCs-D-NSCs , which hints the EB-D-NSCshave more exquisite regulate mechanism than the BMSCs-D-NSCs when the neuralstem cell is induced to differentiate.3, The cell surface grain of the BMSCs-D-NSCs is larger and the surface is rougherthan the one of EB-D-NSCs detected by the AFM,which may have something to dowith its having stronger proliferation ability.Chapter IIIResearch on the differentiation and proliferation of the BMSCs-D-NSCs via theJAK/STAT signal pathwayParti:Influence on the proliferation and differentiation of the BMSCs-D-NSCs in vitroObjective:IL-6 is found to be the important cell factor of JAK/ STAT signal pathway . Manystudies show that it can activate the JAK/ STAT signal pathway in hematopoietic celland the embryonic stem cell. But there is no evidence showing whether IL-6 canpromote the BMSCs-D-NSCs to proliferate and differentiate toward neural cell.Therefore, we intends to detect the expression of STAT3 by the Western- blot methodafter the effect of IL-6 on the BMSCs-D-NSCs ,and make sure the reasonable dosageof IL-6 that can promote the BMSCs-D-NSCs combining to its proliferatedcharacteristics.Methods:1, Induce^ culture and purify the bone marrow neural stem cell:2, Divide into groups:According to the dosage of IL-6: 0 ng/ ml(control), 2 ng/ml, 20 ng/ ml , 100 ng/ ml,200ng/ml.According to the dosage of IL-6R ( the receptor of IL-6): 0+0ng / ml (control),100+100ng / ml (both IL-6 and the receptor of IL-6 are lOOng / ml ), 0+100ng /ml (IL-6 is 0 and the receptor of IL-6 is lOOng /ml), 200+200ng / ml (both IL-6 andthe receptor of IL-6 are 200ng / ml ), 0+200ng ng/ ml (IL-6 is 0 and the receptor ofIL-6 is 200ng / ml)3, Observe the growth status of cell in each group by phase contrast microscope everyday, take a picture in a random visual field and record the experiment result.4, Detect the proliferation of cell applying the alamarblue method: Detect the alamarblue reduction rate of the bone marrow neural stem cell in each group 1,3,5,7 and 9 days later after adding medicine and draw the cell's growth curve .5, Detect the expression of STAT3 by Western blot: Detect the expression of STAT3after different density of IL-6 work on the BMSCs-D-NSCs. Results:1, The IL-6 has the dosage and time dependencity to promote the BMSCs-D-NSCs proliferation;2, The expression of STAT3 in the BMSCs-D-NSCs enhance when the increasement of the dosage of IL-6;3, The eventual density of the IL-6 to improve the efficiency of proliferation of the bone marrow neural stem cell is the 100 ngs/ ml, there are obviously two high peak on the first,third day after giving medicine;4, The proliferation of the BMSCs-D-NSCs can be inhibited by the receptor of IL-6 alone , The proliferation of the BMSCs-D-NSCs can be promoted more obviously by the IL-6 alone instead of combine with the receptor of IL-6, It hints the receptors of IL-6 exist on the surface of the BMSCs-D-NSCs.5, The cell's proliferation can be detected with the alamarblue, It's a simple and effective method to detect the proliferation like stem cell, which is small quantity and need several group of sample for detecting.Part IIEffect on the proliferation and differentiate of the BMSCs-D-NSCs by RapamycinObjective:To observe the effect on the BMSCs-D-NSCs by Rapamycin and make a surereasonable dosage for experiment.Methods:1, Induce, culture and purify the BMSCs-D-NSCs.2, Divide into groups according to the density of RP: 0 ng/ ml (control), 50 ng/ml, lOOng/ml, 200 ng/ml.3, Observe the growth status of cells in each group by phase contrast microscope, record the differentiated rate of cells in reach group.4, Detect the proliferation of cell in each group applying the alamarblue method, and draw the cell's growth curve .5, Detect the expression of STAT3 by Western blot and compare the degree of gray scale.Results:l,The RP can inhibit the differentiation of the BMSCs-D-NSCs , and has dosagedependence;2, The RP inhibits the expression of the STAT3 assosiating with dosage.3,The RP can inhibit the differentiation of the BMSCs-D-NSCs effectively at theeventually density of 100 ng/ ml, it appears obvious inhibition on the first day thatmedicine given.4, The RP inhibits the proliferation but can't promote the differentiation of theBMSCs-D-NSCs , Being independence process of cell the differentiation have itsown control mechanism.Part III:Influence and mechanisms on the proliferation and differentiation of theBMSCs-D-NSCs by JAK/ STAT singal pathway .Objective:To explore the mechanism of the JAK/ STAT signal pathway in the BMSCs-D-NSCsand provide the theory leading for application on it.Methods:1, Induce> culture and purify the bone marrow neural stem cell:2, The experiment is divided into two groups: they are groups of IL-6 and RP , thedensity is 100 ng/ml,we set five time points 0> ^ 24 > 72 > 120 hours after the medicine is given in each group.3, Observe the growth status of cells in each group by phase contrast microscope, record the ratio of differentiated cells in reach group undrer microscope.4, Detect the proliferation of cells applying the alamarblue method.5, Detect the cell's cycle in each group by flow cytometer.6, Detect the expression of JAKl and STAT3 on every time order in each group by Western blot, compare and statistic the degree of gray scale.Results:1, IL-6 whose eventually density is lOOng/ml increase the proliferation index of the BMSCs-D-NSCs to 27.09%, which result in the increasement of the cells in splitting phase (the M phase) and reduction of the cell in GO/ Gl phase The rapamycin whose eventual density is lOOng/ml reduce the proliferation index of the BMSCs-D-NSCs to 15.6%, which result in the reduction of the cells in splitting phase ( the M phase ), increasement of the cell in GO/ Gl phase and increase the apoptosis cell obviously.2, Adding IL-6 whose eventually density is lOOng/ml to BMSCs-D-NSCs,the expression of JAKl reach the peak 24 hours later, and the STAT3 reach the peak 72 hours later, their expression is in accordance with the fastigium of the BMSCs-D-NSCs on the first,third day after adding IL-6.3, Adding RP whose eventually density is lOOng/ml to BMSCs-D-NSCs , the expression inhibition of STAT3 varies with time,it reach lowest lever 24 hours later, the expression of STAT3 is already in accordance with the control group 72 hours later, it hints STAT3 play an important role during the cell proliferation .4, Adding RP whose eventually density is lOOng/ml to BMSCs-D-NSCs, the expression of JAKl has no obvious variety as time going on, there is no marked difference on each time point. This hints the RP affects STAT3.It is not by JAKl butother signal factors to inhibit the proliferation of BMSCs-D-NSCs.Conclusion of chapter III:1, STAT3 is an important factor to affect the proliferation of the BMSCs-D-NSCs in the JAK/ STAT signal pathway. The cell proliferation is enhanced obviously when STAT3 increases expression in BMSCs-D-NSCs. The proliferation is inhibited when it decreases expression in the BMSCs-D-NSCs .2, JAKl shows effec on enhancing the proliferation of BMSCs-D-NSCs via the JAK/ STAT signal pathway ,but shows there is nothing to do with the inhibition of the proliferation about BMSCs-D-NSCs. These hint other factors or other pathway affect the proliferation of BMSCs-D-NSCs.3, STAT3 affects the proliferation of BMSCs-D-NSCs by affecting the cell cycle .4, The JAK/ STAT signal pathway mainly affect the proliferation of BMSCs-D-NSCs, It shows no obvious effect on the differentiation of BMSCs-D-NSCs. To adjust the JAK/STAT pathway can enhance the proliferation of BMSCs-D-NSCs . but the differentiation of BMSCs-D-NSCs have a lot of wok to do.
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