Experimental Research Of Biological Characteristics And Migration Effect Of Bmscs Toward Glioma Cells

Incidence of Glioma is the highest in central nervous system tumors, which is approximately 40 percent. Though the comprehensive treatment can produce some therapeutic effect, the outcome of this malignancy is still not improved drastically. Gene therapy show favourable prospect. But in I and II clinic trial expectant therapy effect was not achieved because of virus vectors's own toxicity, short period of target gene expression, targetin tissue, immunity or applicable vectors. BMSCs as one kind of new vector attract many scientists.Their self-renewal and the multiplex differentiation potential, easily cultured properity, stable exogenous gene expression, safe and reliable resource, avoiding ethics trouble make it possible to carry targeting gene as gene vector. The research of BMSCs biological characteristics including separation, culture, identification and differentiation are base of a large number of BMSCs research, also hot and difficult spot. In vivo BMSCs differentiate into neuron-like cells in very low proportion. It is necessary to increase differentiation ratio into neuron-like cells by appropriate induction strategy. The notable biological effect of bFGF are to induce angiogenesis and increase caryomitosis. BMSCs participate in repairation and integration of the nerve net by differentiation into neuron-like cells limitedly, because in vivo exogenous bFGF are imported in the brain difficultly and endogenous bFGF are activated limitedly. So persistant and steady expression as a result of transfection of related gene could benefit treatment of CNS injury. BMSCs can migrate to injury zone in vivo selectively. This homing ability is the base of oncotherapy. Moreover BMSCs can treat brain tumor through many mechanisms. BMSCs in vitro differ the microenvironment in vivo. Now little workers study that BMSCs in vitro migrate to glioma cell imitating internal environment condition. On account of maneuverability in clinical application in future what kind of BMSCs transplanting pathway would produce the maximum treatment effect on glioma? There are some related factors participating in glioma pathopoiesia, which expression could be regarded as assessment indicator for glioma therapeutic effect. GLUT-3 is regarded as the first rate-limiting factor in brain malignancy glycometabolism. HIF1αparticipate in malignancy angiogenesis, etabolize and apoptosis, which is an important nuclear transcription factor. It has been found little reports about co-expression of GLUT-3 and HIF1αin glioma. For above all, the current study was performed from four aspects as follows.1 Research about BMSCs biological characteristicsObjective To study BMSCs biological characteristics. Methods BMSCs are separated, cultured and induced, subsequently identified by immunohistochemistry, flow cytometry and immunofluorescence. Expression of bFGF vector is structured, sequenced, packed by lentivirus and transfected into BMSCs. In vivo and vitro the proliferation, differentiation and migration of transfected BMSCs are observed by morphology, immunohistochemistry, immunofluorescence, western blot and PCR. Results After BMSCs inoculatin 24h, cell adherence occurred.3d later, BMSCs showed a logarithmic growth. At 1W cell number increased. Their protuberance linked each other and growed slowly. At 12d cells fused and overspreaded bottle. Compared with control group there was significant difference in induction group(P<0.05). CD44,CD29 , CD90,CD105 positive meaned claybank cytoplasm by immunocytochemistry. The positive rate of BMSCs were 92.0±3.2% on CD44,94.6±4.8% on CD29 while CD45, CD11b, CD34 were all negative by immunocytochemistry. The positive rate of BMSCs were 99.64% on CD90,1.23% on CD34,99.54% on CD44,0.43% on CD11b by FCM. After 3d of induction, Nestin was positive. At 7d BMSCs seemed like typical neuron and some from BMSCs showed NeuN and GFAP positive. At early time NSE,NeuN,GFAP positive rate showed an increase with time. After 1W, the rate became lessen. NeuN positive rate in induction group was more than control group(P<0.05). In structuring vector and lentivirus packing tests, SL08 fragment derived from pUC57-SL08 recombinant plasmid screened and restricted were cloned into pLenti6/V5-D-TOPO, then pLenti6- SL08 recombinant plasmid were produced and sequenced. This was a successful structure by sequencing results. The quantitative detection of lentivirus titer was 3×107v.p./ml. 48h later after the specific pLenti6-SL08 fragments targeting bFGF was cloned into rat BMSCs, groups showed an expression of bFGF by Western blot and RT-PCR methods. The expression in BMSCs transfected by bFGF was the strongest. In vitro 24h later after transfection BMSCs number in groups performed an increase.48h or 72h later cell number ascended obviously, cell number in pLenti6-SL08 group were more than other two groups. But no difference about the number in three groups was observed. (P>0.05). At 7d, obvious neuron-like cells were found. At this time the upward rate of cell number dropped, and cell number in pLenti6-SL08 group showed a significant difference compared with other two groups(P<0.05). There was no difference between other two groups(P>0.05). By FCM our results indicated CD90 96.44% , CD34 0.61% in BMSCs-1, CD44 99.95%, CD11b0.73% in pLenti6-SL08 group. 7d later after culture in vitro, in three groups many neuron-like cells were observed, the percent of neuron-like cells in pLenti6-SL08 group was more than other two groups(P<0.05). 48h later after BMSCs transfected by pLenti6-SL08, bFGF expression in transfected BMSCs group was more than in other two groups significantly by western blot and RT-PCR. This transfection was successful. 7d later after culture the expression of Nestin, NeuN and GFAP were observed in three groups by immunohistochemistry. There was an significant difference between Percentage of Nestin and NeuN positive cell in pLenti6-SL08 group and in other two groups(P<0.05). By western blot percentage of Nestin and NeuN positive cell in pLenti6-SL08 group were more than other two groups(P<0.05).There was no difference between other two groups(P>0.05). In cerebral trauma model, immunohistochemistry results indicated that BrdU positive cells at different time points in ipsilateral semisphere are more than in contralateral semisphere(P <0.001). BrdU positive cells at different time points in bFGF transfected group were more than in normal group(P <0.001). 1W later BrdU positive cells number reduce. However, BrdU positive cells in ipsilateral semisphere in bFGF transfected group decreased indifferently (P=0.08). By western blot about bFGF expression there was an obvious difference at different time points in control group(P<0.05). The expression of bFGF in experimental group at 7d increased obviously(P<0.05), decreased insignificantly(P=0.12). The expression of bFGF in control group at 7d increased obviously(P<0.05), decreased significantly(P<0.05). By double-labeling immunofluorescence the co-expression of BrdU and NeuN or GFAP could be observed in two groups. The neuroglial differentiation ratio was more than neural differentiation. In ipsilateral lateral ventricle, cortex zone BMSCs were observed in both groups. There was a significant number difference in two groups(P<0.05). Moreover in ipsilateral lateral ventricle, cortex zone neural differentiation ratio in bFGF transfected groups was more than in control group(P<0.05).Conclusion High purity BMSCs can be produced by separation and culture in vitro. In vivo and vitro bFGF transfected BMSCs can proliferate, differentiate into neuron steady and persistently, and migrate into cerebral trauma location significantly.2 migration effect of BMSCs toward glioma cellsObjective To study migration and depression effect of BMSCs toward glioma cells in vivo and vitro.Methods By immunocytochemistry, western blot whether GLUT-3,HIF-1αcould be one indicator for estimation of glioma biological behavior. Under imitating internal environment condition, migration and depression effect of preprocessed BMSCs toward glioma cells in vitro are assessed by transwell, MTT, plate clone methods. Subsequently, rat C6 model is established. BMSCs are transplanted into the model through two different pathways. Migration, distribution and depression effect of BMSCs toward glioma cells in vivo are examined by behaviouristics, morphology, histopathology and molecular biology. Results By immunocytochemistry the expression of GLUT-3,HIF-1αrelated positively to pathological grades(P <0.001).The results by Western blot were same to by immunocytochemistry. In vitro part: C6 cells increase quickly. After passage growth speed of C6 cells was as before. After co-culture of preprocessed BMSCs and C6 or BMSCs and C6, a large number of cells passed through both indoor membranes by HE. Passed cells in preprocessed BMSCs group were less than in BMSCs group. In the two groups the difference is signigicant(P<0.001). In MTT test, C6 cells were cultured with the supernate from preprocessed BMSCs or BMSCs. C6 cell proliferate more quickly in preprocessed BMSCs group than in BMSCs group(P<0.05). In plate colony test, C6 clones in preprocessed BMSCs group were more than in BMSCs group (P<0.05). GLUT-3 and HIF1αexpressed weaker in preprocessed BMSCs group than in BMSCs group by western blot (P<0.05). In vivo part: Intracranial hypertension symptoms were firstly discovered and most quickly progressed in control group, lastly in BMSCs-1. At 3rd day, the first dead case was found in control group. At 45d, the last dead case was found in BMSCs-1. The survival rate in three groups differed significantly (P<0.05). By HE stain, tumor cells were intensive, infiltrated normal brain tissue and made a distinction between normal brain tissue. Tumor cells showed small volume, big nucleolus, nuclear division and obvious nests. Tumor volume in control group was biggest, least in intraventricular injection group(P<0.05). By BrdU immunohistochemistry stain it has been testified that BMSCs distributed vessel, hippocampus and ventricle circum obviously after BMSCs transplanted into rat C6 model from contralateral lateral ventricle. Distribution of BMSCs in the ipsilateral hemisphere were more than in the tumor(P <0.001). Distribution of BMSCs in the ipsilateral hemisphere were more than in contralateral hemisphere(P <0.001). BMSCs number in the tumor in intracerebroventricular injectjion group were more than in intravenous injection caudal vein group (P <0.001). BMSCs number in the ipsilateral hemisphere in intracerebroventricular injectjion group were more than in intravenous injection caudal vein group (P <0.001). The results in contralateral hemisphere were the same to the previous(P <0.001). By Western blot in BMSCs-1, BMSCs-3 and control group expression of GLUT-3 and HIF1αincreased by degrees respectively(P<0.05). Just like results of Western blot expression of GLUT-3 and HIF1αalso increased by degrees respectively(P<0.05). Conclusion It has been tested elementally that GLUT-3 and HIF1αas important factors participate in glioma pathogenesis, and reprocessed BMSCs show stronger taxis, suppress glioma proliferation and downregulate the expression of GLUT-3 and HIF1α. BMSCs in intracerebroventricular injectjion group show stronger taxis, suppresse neoplasm, downregulate the expression of GLUT-3 and HIF1αand improve prognosis sigjificantly.