The Role Of Basic Fibroblast Growth Factor And Vitamin C On The Proliferation Of Bone Marrow Mesenchymal Stem Cells

ObjectiveBone marrow mesenchymal stem cells ( MSCs) , reside in bone marrow mesenchyme, is a stem cell that be able to differentiate into several mesenchymal lineages. At present, MSCs have been transplanted to restore central lesion and to treat various nervous system disease. This has become a hot spot of neu-roscience research. But there are few MSCs in adult marrow. It has been observed that after MSCs reach the first confluence in vitro, their proliferation rate is reduced and it is associated with loss of multipotentiality. These confined MSCs to make use of cell and gene therapy.The purpose of this study is to establish an ideal and effective proliferation method for MSCs with vitamin C and bFGF. We infuse the MSCs into cerebral infarction rat to examine the growth behavior of cells. This study provides a new idea of acquiring adequacy biologic activity MSCs to treat nervous system disease.Methods1. MSCs were isolated culture and purify, the second - generation cells were used for experiment. They were divided into four groups according to adding material (group 1: control group; group 2: MSCs cultured with vitamin C; group 3: MSCs cultured with bFGF; group 4: MSCs cultured with bFGF andvitamin C). Observe proliferation and morphological behavior of cultured MSCs with phase contrast microscope.2. Identification of cells origin; cells were stain for CD34,CD44 and CD29 with immnohistochemistry to identify origin of cells.3. MSCs of each group were seeded and cultured into 24 and 96 shadow masks and the growth curve of MSCs was drawn. MTT colorimetric method was used to assay numeric value and to estimate vital force of culture cells.4. Culturing after 96, the acting growing MSCs of each group were detached by 0.25% trypsin and fixed with 4℃ ethanol for 30 minutes in suspension, washed 3 tines with ice cold PBS, treated with RNAase 1mg/ml for 30 minutes in 37℃, centrifuged down, resuspended in lOmg/ml propidium iodide for 15 minutes in dark 4℃, then passed through flow cytometer.5. MSCs of Vc + bFGF and control groups were labeled by Brdu and infused into cerebral infarction rat to examine the growth behavior of cells.6. At 2 weeks postimplantation, corona - cut rat brains after paraffin embedding, serial sections 8μm thick were cut and stained with either hematoxylin and eosin or Brdu tissue stain for immunohistochemical studies and examined the responses of tissue. Observe three fields of vision at each section. Calculate the average number of brdu - positive cells in the sections to analysis.Results1. Morphology observation: Primary culture of MSCs: At the 1-2 days, MSCs start to attach to the bottom and became fusiform shape. After 1 weeks, it is clear seen that CFU - F (colony forming unit — fibroblastic) form. About 14 days,there is over 80% cells fusion. Serial subcultivation of MSCs: Obversing the growth behavior of MSCs that cultured with factors, cells attach to the bottom after 24 hour . The cells of fourth group grow fastest and 80% cells fuse at 4th day. The cells of control group growth slowly, and 80% cells didn t fusion after 7 days.2. Identification of cells origin; All of culture MSCs are positive stained by CD44 and CD29 antibody. The cells are negative stained by CD34 antibody.3. The growth curve of MSCs: At the first day the cells proliferated slowly. This period showed as incubation period corresponding to curve. The second day after seeding, the MSCs of group 2,3,and 4 proliferate obviously, cell number increased quickly and came into exponential fashion. After that, cell number plateau as growth ceased until the 5th day. Comparing with groups 2,3and 4, the growth curve of the control group moved to right and cell number increased slowly over the first 4 days and then in rapid, exponential phase of growth began.4. The effects of Vc and bFGF to the proliferation of MSCs: MTT colori-metric method estimated vital force of culture cells shows: Comparing with control group , there is significant increase in the groups2,3and 4 ( P < 0. 05 ). At the 7 day, the proliferation of groups2,3 and control cells begin to decrease, but the MSCs that cultured with Vc + bFGF still to remain with quick proliferating.5. The cell cycle examination of MSCs: flow cytometer analysis shows that compared with control group, the percent of cells in S + G2 + M phase which cultured with factors increased obviously ( P < 0. 01 ) but the cells in G0 + G1 phase decreased. In the Vc + bFGF group, the percent of cells in S + G2 + M phase is the biggest compared with the others(P <0.01).6. Model building resultant HE dyeing: the animals of two groups appeared some symptom after completely analepsia:the left forefoot can't stretch and body circling to left. These display that model building is succeed. HE dyeing display block focus tissue looseness, colour pale, boundary distinct. The spaces of nerve cells widen in foucus.7. Immunohistochemistry assessment of BrdU: there are mostly BrdU positive stained cells in ischemic hemisphere and few in opposite side. BrdU positive stained cells assume dark brown. The average number of positive cells in ischemic hemisphere; control group is 55.2 24. 8/section; VC + bFGF group is 53.6 10.7/section(P>0.05).