Protective Effect Of Mesenchymal Stem Cells To 1-Methyl 4-Phenylpyridine Induced Apoptosis In PC12 Cells

Protective Effect of Mesenchymal Stem Cells to 1-Methyl 4-Phenylpyridine-Induced Apoptosis in PC12CellsObjectiveRecently study discovered, Bone marrow stromal cells(BMSCs), or be called mesenchymal stem cells(MSCs) have stem-cell character and the potential for multidirectional differentiation, MSCs is easy to get, develop and propagate in vitro. Autotransplanting avoids immune rejecting reaction too. As a result, mesenchymal stem cells have vast of applied foreground as the cytology treatment foundation used for the central nervous system disease and its trauma repair. Parkinson disease(PD) is a kind of chronic progress nervous system recessive disease. The main "pathologic characteristic is the dopaminergic neuronal recession in substantia nigra striatum. The morphological shows dopaminergic neuron(DA) presents the apoptosis kind change in PD patient's brain, which prompt cell apoptosis may participate the pathogenesis process of the PD. 1-methyl 4-phenyl pyridium(MPP⁺) can injure the DA optionally. The pheochromocytoma(PC12) cells are the cells of addicted chrome cell tumors for the big rat adrenal glands, its receivers and transmitters are similar to DA, they can illuminate consistent problem with original generation nerve cell, so PC12 cell with MPP⁺ model is often the cell model by way of studying PD. The MSCs can secrete cell factors considered to be connected with they having the protective function to the DAs. Their conditional culturable liquid was cultured, pured and collected in this trial, examining secreted cell factors by MSCs(GDNF, BDNF, IL-6) that have the protective function to the DAs. To investige the effect of the mesenchymal stem cells(MSCs) secretary factors to 1-methyl-4-phenyl pyridium ion(MPP⁺) induced apoptosis in PC12 cells by evaluating MSCs secretary factors influencing to c-Jun, bcl-2 and caspases3 of the MPP⁺ induced PC12 cells.Methods1. The collections and the inspissation of the original MSCs supernatant2. MSCs surface antigen measurement(immunocytochemistry)The first antibody is the CD44, CD45 and the second antibody is CY3.3. Measurement the protein level of GDNF, BDNF and IL-6 of MSCs secreting neurotrophic factor(ELISA)4. Culture and medical management of PC12 cellsPC12 were disparted into 4 group: control group, it is do not joined any medicine in the cell culture system; simple supernatant of MSCs group, it is joined the supernatant of MSCs in the cell culture system after inoculating 24h, the concentration is 30μ, 60μl and 120μl; MPP⁺ group, it is joined the MPP⁺ in the cell culture system after inoculating 24h and eventual concentration is 100μmol·L-1, 200μmol·L-1 and 400μmol·L-1 respectively; unite group, it is joined the 30μl, 60μl and 120μl supernatant of MSCs after inoculating 24h following joining the 200μmol/L MPP⁺. Examination of the following index after joined MPP⁺ 24h and 48h.5. The measurement of cell vitality(MTT)6. The morphological changes of apoptosis were observed under transmitting electron microscopy(TEM)7. Apoptosis ratio was analyzed by PI dyeing Flow Cytometric(FCM)8. Measurement of the expression of the protein of bcl2, tyrosinehydroxylase (TH), caspase3 and c-Jun(immunocytochemistry)9. Measurement of the expression of the mRNA of bcl-2, TH, caspase3 and c-Jun (RT-PCR) The cells were managed give MSCs by the pure MSCs supernatant and MPP⁺ and distilled total RNA with Trizol. PCR expand with cDNA as template after RT reaction was over. Each purpose gene and the NADPH were all taken 5μl after the expanding reaction was over, electrophoresis examination was carried on the 1.5% agar sugar gel, the gel picture was analyzed by gel picture analysis instrument. Express level of bcl2, c-Jun, caspase3 and TH mRNA was carried on with half quantitative analysis according ratio of gel stripe density of the purpose gene and inside consultation.Results1. MSCs may separate and proliferate in vitro, were positive for CD44 and negative for CD45(Fig1, 2, 3, 4).The cells stuck the wall were spindle-shaped, single or a few cells clones and uniform. Culture 2～5d were the growing latent period, and proliferation of the cells grew slowly, the variety of the cell numbers was not obvious. Culture 6～12d were the quickly proliferate period, and the arrangement of the cells have certain direction, the growing shape of the cells was gyrate. Culture 15～18d, 80%～90% cells emerged fusion, the cells overlapped between clones, and have no the phenomenon of contacting control. The cells to be digested by trypsin completely stuck the wall within 12h, stretched and changed into spindle-shaped renewedly, and kept the appearance of the original generation cells, grew quickly, attained the complete fusion after 7～10d. MSCs were positive for CD44 is the proof of stem cells and negative for CD45 is not the proof of hematopoietic cells by immunocytochemistry (Fig1A, B, C, D).2. The culture supernatant of MSCs can secrete the neurotrophic factor of GDNF, BDNF, IL-6(Tablel).3. The results of MTT showed the secretary factors of MSCs can advance the cell viability and resist the toxic function of MPP⁺ to PC12 cells.100μmol·L-1, 200μmol·L-1 and 400μmol·L-1MPP⁺ managed group after inoculating 24h, the cell viability were lower to 0.76±0.11, 0.47±0.12 and 0.34±0.10 respectively than that of the control group(0.97±0.02); After inoculating 48h, the cell viability were lower to 0.61±0.01, 0.30±0.04 and 0.20±0.04 respectively than that of the control group(0.94±0.05). The more the concentration was, the more the toxicity was, the neighbor concentration of the difference have notable significance(P＜0.05); the more the time acted, the more the toxicity was to PC12 cells in the same concentration(P＜0.05) (Fig2A, B).Comparing with 200μmol·L-1MPP⁺ managed group after inoculating 24h, the cell viability of the unite group(the concentration of MSCs secretary factors are 30μ1, 60μ1, 120μ1 respectively) were higher to 0.67±0.13, 0.77±0.20 and 0.85±₀.11; After inoculating 48h, the cell viability were higher to 0.50±0.11, 0.65±0.05 and 0.75±0.05. The more the concentration was, the more the cell viability was, the neighbor concentration of the difference have notable significance(P＜0.05) (Fig2C).4. TEM can observe the apoptosis of the PC12 cells.We can observe the symptom of apoptosis of decreased cellular protruding, small ratio of nucleus/cytoplasm, increased space in cytoplasm, increased gap of periphery in nucleus uncertainly, increased poikilochromatin collected in edge, and the incomplete cell membrane(Fig3B).5. The results of FCM showed that secretary factors of the MSCs can decrease the ratio of apoptosis of the PC12 cells treated with MPP⁺.100μmol·L-1, 200μmol·L-1 and 400μmol·L-1MPP⁺ managed group after inoculating 24h, the ratio of apoptosis were higher to (27.76±1.45)%, (42.34±3.21)% and (56.63±4.01)% respectively than that of the control group(1.51±0.11)%; After inoculating 48h, the ratio of apoptosis were higher to (43.78±3.29)%, (55.36±3.98)% and (79.35±6.25)% respectively than that of the control group(1.54±0.18)%. The ratio of apoptosis of the PC12 cells increased in file along with the increase of concentration of secretary factors of MPP⁺(P＜0.05) (Fig4A, B); Comparing with 200μmol·L-1MPP⁺ managed group after inoculating 24h, the ratio of apoptosis of the unite group(the concentration of MSCs secretary factors are 30μ1, 60μ1, 120μ1 respectively) were lower to (31.96±2.89)%,(17.89±1.78)% and (10.08±0.91)%; After inoculating 48h, the ratio of apoptosis were lower to (39.43±3.08)%, (23.13±2.18)% and (14.21±1.41)% (P＜0.05). The ratio of apoptosis of the PC12 cells of different concentration of secretary factors of unite group have notable difference, the neighbor concentration of the difference have notable significance (P＜0.05) (Fig5A,B).6. The results of immunocytochemistry showed the content, of protein of TH and bcl-2 in the unite group was greater than the MPP⁺ group, while caspase3 and c-Jun was lower than the MPP⁺ group.Positive cell number of bcl2 and TH obviously increase of 120μl secretary factors of MSCs unite group comparing MPP⁺ managed group (Fig6,7); positive cell number of c-Jun and caspase3 obviously decrease of 120μl secretary factors of MSCs unite group comparing MPP⁺ managed group (Fig8,9).7. The results of RT-PCR showed the content of mRNA of TH and bcl2 in the group of secretary factors of MSCs was greater than the MPP⁺ managed group, while caspase3 and c-Jun was lower than the MPP⁺ managed group.After inoculating 24h and 48h, the ratio of the content of bcl2 and THmRNA with GADPHmRNA of the unite group (the concentration of MSCs secretary factors are 30μl, 60μl, 120μl respectively) were higher to 0.63±0.02, 0.71±0.02, 0.85±0.01 (24h), 0.49±0.01, 0.57±0.01, 0.67±0.01 (48h) and 0.55±0.02, 0.62±0.01, 0.69±0.01 (24h), 0.43±0.02, 0.52±0.01, 0.60±0.01 (48h) respectively than that of the 200μmol·L-1 MPP⁺ managed group in the same time (bcl2 and TH were 0.57±0.01 (24h), 0.36±0.01 (48h) and 0.44±0.01 (24h), 0.28±0.01 (48h) respectively). The content of mRNA of TH and bcl2 increased in file along with the increase of concentration of secretary factors of MSCs; the neighbor concentration of the difference have notable significance(P＜0.05) (Fig10A, B and 11A,B). After inoculating 24h and 48h, the ratio of the content of c-Jun and caspase3mRNA with GADPHmRNA of the unite group (the concentration of MSCs secretary factors are 30μl, 60μl, 120μl respectively) were lower to 0.59±0.01, 0.52±0.02, 0.43±0.01 (24h), 0.68±0.02, 0.60 ±0.01, 0.52±0.01(48h) and 0.59±0.01, 0.53±0.01, 0.44±0.03(24h), 0.63±0.01, 0.56±0.01, 0.50±0.01(48h) respectively than that of the 200μmol·L-1 MPP⁺ managed group in the same time(c-Jun and caspase3 were 0.70±0.02(24h), 0.75±0.01(48h) and 0.76±0.01(24h), 0.714±0.01(48h) respectively). The content of mRNA of c-Jun and caspase3 decreased in file along with the increase of concentration of secretary factors of MSCs; the neighbor concentration of the difference have notable significance(P＜0.05) (Fig10C, D and 11C, D).ConclusionAccumulating evidence indicates that the differentiation potential of pluripotent MSCs is not restricted to lineages found in the tissue where they originate and MSCs can give rise to central nervous system cells. MSCs become the most attractive and ideal treatment tool because MSCs is easy to separate and culture and have high proliferation. One difficult problem of MSCs is there is not particular cell mark of MSCs to be discovered. Current generally accepted and opposite particular cell marks of MSCs are CD90, CD71 and CD44 antigen, but have no the CD34, CD45 and CD11b antigen which is the mark of hematopoietic stem cell. Cells surface antigen CD44 and CD45 were measured by immunocytochemistry, MSCs of the experiment needed were positive for CD44 and negative for CD45. The protect function of MSCs to DA is considered to be relevant with the cell factor that MSCs secrete, which is the one of the mechanism of reducing neuronal apoptosis. Under the physiological state, MSCs can secrete various growth factors of the NGF, VEGF, GDNF, BDNF, bFGF, HGF, CSF 1 and cytokine of the IL-6, IL-7, IL-8, IL-11 and etc; These neurotrophic factors may exert the neural protect function and reduce neuronal apoptosis by the different mechanism. A mechanism of MSCs treating PD is MSCs can stimulate to produce various neurotrophic factors. Secretary factors of MSCs can secrete neurotrophic factors of GDNF, BDNF and IL-6 by ELISA method in this experiment. MSCs secrete neurotrophic factors of GDNF, BDNF and IL-6 and exert the neural protect function to damaged DA by MPP⁺. Particularly the neurotrophic factor of GDNF is generally accepted to promote survivance DA in midbrain. GDNF can protect and repair the growing and mature DA, can strengthen ingestion to dopamine of cultured DA in vitro and promote the differentiation of DA. BDNF have trophic function to nigrat DA and aminobutyric acidgic neuron of cultured embryo basal forebrain. IL-6 act on DA, strengthen the activity of tyrosinehydroxylase, extent existent time of neuron, and increase endocellular dopamine. The secretary factors of MSCs can protect the toxic injury, and the higher the concentration was, the better protect function of the secretary factors was by MTT method; FCM consequence register that the MSCs can obviously alleviate the inducing apoptosis of function of MPP⁺ to the PC12 cells; their alleviative function of MPP⁺ to the PC12 ceils increased along with the increase of concentration of secretary factors of MSCs. Parkinson disease(PD) is a kind of chronic progress nervous system disease. The pathogenesis of PD is a hotspot that people research and pay attention to. PD is connected with DA cellular apoptosis in recent years along with the advance of neurobiochemistry and histochemistry. Internal cell regulation of nigral DA cellular apoptosis is a very complicated process, ROS, bcl2, bax and caspases3 all act on important role, its nuclear tache may be the toxin of inside and outside environment broke the calcium balance, resulted in the overload of calcium and respirational chain producing the free radicals to decrease bcl2, activate caspases3 in order to commonly participate in regulating cellular apoptosis and cause DA cellular apoptosis finally.The results of immunocytochemistry showed the secretary factors of MSCs can obviously increase the expression of the anti-apoptosis factor bcl2 and the MSCs can obviously alleviate the inducing apoptosis of function of MPP⁺ to the PC12 cells; The results of PT-PCR showed the secretary factors of MSCs can obviously increase the content of the anti-apoptosis factor bcl2 and alleviate the inducing apoptosis of function of MPP⁺ to the PC12 cells; their alleviative function of MPP⁺ to the PC12 cells increased along with the increase of concentration of secretary factors of MSCs. Bcl2 is membranous conformity protein and exists in litosone, nucleus membrane, endoplasm of cells. The research expressed the protect function of bcl2 protein to nigral DA by anti-oxygenation. Our research showed MSCs can obviously improve the decrease of bcl2 protein and mRNA damaged by MPP⁺ and exert nervous function. The results of immunocytochemistry showed the secretary factors of MSCs can obviously decrease the expression of the stimulative apoptosis factor c-Jun and caspase3 and the MSCs can obviously alleviate the inducing apoptosis of function of MPP⁺ to the PC12 cells; The results of PT-PCR showed the secretary factors of MSCs can obviously decrease the content of the stimulative apoptosis factor c-Jun and caspase3 and alleviate the inducing apoptosis of function of MPP⁺ to the PC12 cells; their alleviative function of MPP⁺ to the PC12 cells increased along with the increase of concentration of secretary factors of MSCs. Caspase is a effectible implement of cellular apoptosis, caspase3 is especially thought the pathway of certain pass of cellular apoptosis continuous reaction. The losing of nigral DA in midbrain is the most pathologic characteristic of PD. The losing of nigral DA in midbrain is positively connected with caspases3 in obituary PD patients by immunohistochemistry. Our research showed MSCs restrained course of cellular apoptosis by interdicting caspases3 activation. Already certainly former apoptosis signal numerator JNK(c-JunN terminal stimulating enzyme) is connected with nervously degenerative disease. JNK signal continuous reaction may be one of the mechanism which MPTP induced the death of neuron. And start cellular apoptosis transmit system by c-Jun. The protect function of MSCs to DA is connected with restaining JNK activity, reducing c-Jun phosphatization.MSCs may rapidly proliferate in vitro, were positive for CD44 and negative for CD45. MPP⁺ can induce apoptosis in PC12 cells. MSCs have potencial in the secretion neurotrophicfactors of GDNF, BDNF, IL-6 and so on to benefit for recessive DA, so MSCs may exert a protective effect against MPP⁺ induced apoptosis in PC12 cells. Its strong and weak was connected with different concentrations. Its inhibiting apoptosis mechanism may be achieved by upregulating bcl2 or deregulating caspase3 and c-Jun various pathways. The research provided definitely experimental basis to PD's treatment.