Differentiation Of Bone Marrow Mesenchymal Stem Cells Into Osteoblasts Induced By Echinacoside And Its Mechanism

Purpose:The research based on traditional Chinese medicine theory "kidney controlling bone" and the BMP2 -Smad-Runx2 signaling pathways of osteogenetic differentiation, and cell culture, Elisa detection, immunofluorescence staining, western blot, rt-pcr technique and so on were used to observe the effect of echinacoside serum which belongs to active ingredients of cistanche desertiola(a kind of traditional Chinese medicine to invigorate the kidney) on the osteogenic differentiation in bone marrow mesenchymal stem cells, and revealed mechanism of inductive effect of echinacoside serum at cellular and molecular levels. The study want to provide a new inducer for sources of seed cells of bone tissue engineering, and provide basis theory of bone marrow mesenchymal stem cells in tissue engineering、fracture healing and bone defect repair. Moreover, the research would provide a new train of thought for the further development and utilization of echinacoside.Material and method:Part 1:1. Preparation of echinacoside serum.60 SPF SD rats were divided into normal controlled group and echinacoside (Echinacoside, ECH) group by the random number table method. Rats in echinacoside group were given echinacoside powder aqueous solution(the concentration of echinacoside was 4 mg/ml, each rat was gavaged lml every time), such as normal control group were given same volume of physiological saline, two groups were gavaged 2 times a day for 4 days. After the last gavage, all rats would be injected 10% chloral hydrate anesthesia into abdominal cavity, and blood was collected from the abdominal aorta with negative pressure, made the blood coagulation naturally at room temperature, serum was separated after centrifugal separation, inactivated in 56℃ water bath for 30 min, subpackaged the serum into aseptic centrifuge tube after filtrated and sterilized, and reserved in -80℃ refrigerator at last.2. Separation and culture of bone marrow mesenchymal stem cells (BMSCs). Primary BMSCs were separated by whole bone marrow adherent screening method, and then cultured primary BMSCs in saturated humidity incubation box with 37℃ and 5% CO2. Observed by Inverted microscope in the 3rd day, changed half of culture medium if BMSCs grew with static adherence. Afterward, changed culture medium once every 2 days. Passaged cells when cell fusion of primary BMSCs was up 80% in the 14th day.3. Identification of BMSCs. P3 generation of BMSCs, vaccinated in the 24 hole culture plate with cover glass, put culture plate into incubation box with 37℃ and 5% CO2 for 4 days, replaced the medium every day. Afterwards, immunofluorescent staining was used to detect expression of CD29, CD34, CD44. 4. Influence of echinacoside serum on the proliferation in BMSCs. BMSCs of P3 generation which were in the logarithmic phase period, were adjusted with it concentration of 3 x 104/ml and vaccinated in 7 pieces of 96 well culture plate, sterile PBS were filled in edge holes of culture plate, and 100ul per cell suspension were in the rest of the holes. Following, cultured in incubation box with 37℃ and 5% CO2. Next day, divided BMSCs into normal control group, the positive control group and the different concentration of echinacoside serum groups (2.5%,5%,7.5%,10%,15%,20% medicated serum) replaced different corresponding medium, each group with six holes were set, the medium were daily replaced at the same time, and culture would be lasted 7 days. Take 1 piece of culture plate every day, 20ul MTT solution (5 mg/ml) was added in every hole, incubated for 4 hours, discarded medium, DMSO was added in every hole and low shocking for 10 min to dissolved the crystallization fully.Following, microplate reader was used to detect absorbance of each hole with 490 nm wavelength. At last, drawing curve of cellular proliferation.The second part:1. Morphologic detection of alkaline phosphatase when echinacoside induced BMSCs to osteogenetic differentiation. BMSCs of P3 generation which were in the logarithmic phase period, were vaccinated in the 24 hole culture plate with cover glass, put culture plate in incubation box with 37℃ and 5% CO2, Next day, divided BMSCs into different experimental groups (same as part1), replace different culture medium, each group with three holes were set, replaced medium once every 2 days, and culture would be lasted 7 days. Following,4% paraformaldehyde was used to fix cells for 30 min, cells were incubated with fresh application liquid substrates at 37℃ and without light, after 15 min, washed cells with distilled water, observed with the microscopy at last. 2. Detection of alkaline phosphatase content in cell lysis supernatant when echinacoside induced BMSCs to osteogenetic differentiation. BMSCs of P3 generation which were in the logarithmic phase period, were adjusted with it concentration of 3 x 104/ml and vaccinated in the 24 hole culture plate, put culture plate into incubation box with 37℃ and 5% CO2,Next day, divided BMSCs into different experimental groups(same as part1), replaced different culture medium, each group with three holes were set,. replaced medium once every 2 days, and culture would be lasted 7 days. Following, discarded medium and washed twice with PBS.500μl RIPA was added in every hole to dissociate cells fully. And then, moved the samples into EP tube, used ultrasonic cell disruptor with 3000rpm and 20 min, collected the supernatant and put it into -20℃ refrigerator. According to the ELISA kit instructions, measured content of ALP of each sample.3. Detection of osteocalcin by immunofluorescence staining when echinacoside induced BMSCs to osteogenetic differentiation. BMSCs of P3 generation which were in the logarithmic phase period, were vaccinated in the 24 hole culture plate with cover glass. Next day, divided BMSCs into different experimental groups(same as partl), replaced different culture medium, each group with three holes were set, replaced medium once every 2 days, and culture would be lasted 14 days. And then, immunofluorescent staining was used to detect expression of osteocalcin. 4. Detection of osteocalcin content in cell lysis supernatant when echinacoside induced BMSCs to osteogenetic differentiation. BMSCs of P3 generation which were in the logarithmic phase period, were adjusted with it concentration of 3 x 104/ml and vaccinated in the 24 hole culture plate, put culture plate into incubation box with 37℃ and 5% CO2, Next day, divided BMSCs into different experimental groups(same as partl), replaced different culture medium, each group with three holes were set, replaced medium once every 2 days, and culture would be lasted 14 days. Next day, RIPA was used in every hole to dissociate cells fully. And then, according to the ELISA kit instructions, measured content of BGP in each sample.5.Detection of collagen I by immunofluorescence staining when echinacoside induced BMSCs to osteogenetic differentiation. BMSCs of P3 generation which were in the logarithmic phase period, were vaccinated in the 24 hole culture plate with cover glass, put culture plate into incubation box with 37℃ and 5% CO2, Next day, divided BMSCs into different experimental groups (same as partl), replaced different culture medium, each group with three holes were set,. replaced medium once every 2 days, and culture would be lasted 7 days. And then, immunofluorescent staining was used to detect expression of collagen I.6.Detection of mineralized nodule staining when echinacoside induced BMSCs to osteogenetic differentiation. BMSCs of P3 generation which were in the logarithmic phase period, were adjusted with it concentration of 3 x 104 /ml and vaccinated in the 24 hole culture plate, put culture plate into incubation box with 37℃ and 5% CO2, Next day, divided BMSCs into different experimental groups (same as partl), replaced different culture medium, each group with three holes were set,. replaced medium once every 2 days, and culture would be lasted 21 days. And then, discarded medium, 4% paraformaldehyde was used to fix cells for 30 min, washed cells three times with PBS, stained with 0.1% alizarin red S (pH8.3) for 30 min with 37℃, washed several times with PBS, after natural drying, observed with the microscopy.part 3:investigation on mechanism of echinacoside serum on inducing osteogenic differentiation in BMSCs.1. Detection of expression of BMP2 when echinacoside induced BMSCs to osteogenetic differentiation. BMSCs of P3 generation which were in the logarithmic phase period, were vaccinated in the 24 hole culture plate, put culture plate into incubation box with 37℃ and 5% CO2, Next day, divided BMSCs into different experimental groups(normal controlled group、positive control group、 echinacoside group), replaced different culture medium, each group with three holes were set, replaced medium once every 2 days, and culture would be lasted 7 days. Following, immunofluorescence staining、western blot、 RT-PCR technique were used to detect expression of BMP2.2. Influence of noggin which was inhibitor of BMP2 on content of alkaline phosphatase in cell lysis supernatant when echinacoside induced BMSCs to osteogenetic differentiation. BMSCs of P3 generation which were in the logarithmic phase period, were adjusted with it concentration of 3 x 104/ml and vaccinated in the 24 hole culture plate, put culture plate into incubation box with 37℃ and 5% CO2, Next day, divided BMSCs into different experimental groups(normal controlled group、positive control group、 echinacoside group、Noggin which was inhibitor of BMP2 group),replaced different culture medium, each group with three holes were set,replaced medium once every 2 days,and culture would be lasted 7 days.Dssociated cells fully in the 8th day, collected the supernatant and put it into -20 ℃ refrigerator. According to the ELISA kit instructions, measured content of ALP of each sample.3. Influence of noggin which was inhibitor of BMP2 on expression of Smad1/5/、ZHX3、Runx2 and OSX. BMSCs of P3 generation which were in the logarithmic phase period, were vaccinated in the 24 hole culture plate with cover glass, put culture plate into incubation box with 37℃ and 5% CO2, Next day, divided BMSCs into different experimental groups (normal controlled group、positive control group、echinacoside group、Noggin which was inhibitor of BMP2 group), replaced different culture medium, replaced medium once every 2 days, and culture would be lasted 7 days. And then, immunofluorescence staining、 western blot、RT-PCR technique were used to detect expression of Smadl/5/8、ZHX3、Runx2、mRNA and protein of OSX.Results:1. Observation of morphological characteristics of BMSCs which were cultured by bone marrow adherent method. The original generation BMSCs were cultured 1 day, adherent cells were less, shapes of cells were approximatly circle. The number of spindle adherent cells gradually increased after 2-3 day, cells became longer. Number of spindle adherent cells were further increased in the 5th day, growth of cells began to form the scattered small colony and chrysanthemum pattern. In the 10th days, cell colony were gradually bigger and increased, cellular density of the colony center was enhanced, the cellular volume was relatively small, cells around the colony were less and were radially distributed.sizes of cell colony were different and fused each other. Degree of fusion close to 80% in the 14th day, subcultured the cells after using trypsin digestion, and then, shapes of cells became gradually uniform, P3 generation of BMSCs characterized by fibroblast, shapes of cells were spindle, cells showed the polar and spiral growth.2. Identification of cells which were cultivated by the whole bone marrow adherent method. Shape of P3 generation cells were in accord with the characteristics of BMSCs. Expression of CD29、CD34、CD44 on cellular surface had be detected by immunofluorescence staining. The result showed that expression of CD44, CD29 were positive, expression of CD34 was negative positive, it was demonstrated that cells were cultivated by whole bone marrow adherent cells had the characteristics of BMSCs, and could meet the needs of the experiment.3. Effect of echinacoside serum on proliferation of BMSCs. Used echinacoside serum with different concentration to culture BMSCs, and then, detected by MTT test. At the same time to compared with normal control group,2.5% echinacoside serum group could not promote the proliferation of BMSCs, no significant difference (P>0.05),7.5%、10%、 15% echinacoside serum groups could promote proliferation of BMSCs significantly (P<0.05),15% echinacoside serum had the strongest effect on promoting proliferation of BMSCs, with significant difference (P< 0.01). If drug concentration was too high, such as 20% echinacoside serum group had no effect on proliferation of BMSCs. From the angle of promoting cellular prol iferation, concentration of 7.5%,10%,15% echinacoside serum groups were considered to be one of the best effect.4.Change of alkaline phosphatase during the process that echinacoside serum induced osteogenetic differentiation of BMSCs. Cultured cells for 7 days with different treatment factors, detected the change of ALP content in the cell lysis supernatant fluid by ELISA. Compared with the normal control group, expression of ALP in positive control group and 7.5%,10%,15% echinacoside serum groups increased significantly, there were significant difference (P<0.05). Compared with the positive control group, expression of ALP in 7.5% and 10% echinacoside serum groups was obviously higher, there were significant differences (P<0.01). Results of ALP staining showed that tan granules were observed in the cells were less in the normal control group and 2.5% echinacoside serum group, the two groups had no significant difference (P>0.05); Compared with normal control group, tan granules were observed in the cells were obviously increased in the positive control group and 7.5%、10%、15% echinacoside serum groups, there were significant differences (P<0.01); Compared with positive control group, there were no significant difference (P<0.05) in expression of ALP in 10% echinacoside serum group; Expression of ALP is slightly increased in 20% echinacoside serum group, however, there were no significant difference (P>0.05) compared with that of positive control group.5.Change of osteocalcin content during the process that echinacoside serum induced osteogenetic differentiation of BMSCs. Cultured cells for 14 days with different treatment factors, detected the change of BGP content in the cell lysis supernatant fluid by ELISA, compared with the normal control group, expression of BGP in positive control group and 10% echinacoside serum group increased significantly, there were significant difference (P< 0.05).Compared with the positive control group, expression of BGP in 10% echinacoside serum group was obviously higher, there was significant differences (P< 0.05). Results of immunofluorescence staining showed, compared with the normal control group, expression of BGP in positive control group and 10% echinacoside serum group increased significantly, there were significant difference (P<0.01). Compared with the positive control group, expression of BGP in 10% echinacoside serum group was obviously higher, there were significant differences (P<0.05).6. Change of collagen I during the process that echinacoside serum induced osteogenetic differentiation of BMSCs. Cultured cells for 7 days with different treatment factors, result of immunofluorescence staining showed, compared with the normal control group, expression of collagen I in positive control group and 10% echinacoside serum group increased significantly, there were significant difference (P<0.01). Compared with the positive control group, expression of collagen I in 10% echinacoside serum group was obviously higher, there were significant differences (P<0.05).7. Detection of mineralized nodule during the process that echinacoside serum induced osteogenetic differentiation of BMSCs. Cultured cells for 28 days with different treatment factors, result of alizarin red staining showed, compared with the normal control group, the number of mineralized nodule in positive control group and 10% echinacoside serum group increased, there were significant difference (P<0.01). Compared with the positive control group, the number of mineralized nodule in 10% echinacoside serum group was obviously increased, there were significant differences (P<0.05).8. Detection of expression of BMP2 during the process that different concentrations of echinacoside serum induced osteogenetic differentiation of BMSCs. Compared with the normal control group, expression of mRNA and protein of BMP2 in positive control group^ echinacoside group increased obviously, there were significant difference (P< 0.01). Compared with the positive control group, expression of mRNA and protein of BMP2 in echinacoside group increased obviously (P<0.01).9. Influence of noggin which was inhibitor of BMP2 on content of alkaline phosphatase in cell lysis supernatant when echinacoside induced BMSCs to osteogenetic differentiation. Cultured cells for 7 days with different treatment factors, ELISA was used to detected content of ALP in the cell lysis supernatant fluid. Compared with the normal control group, expression of ALP in positive control group and echinacoside serum groups and noggin which was inhibitor of BMP2 group increased significantly, there were significant difference (P<0.05). Compared with the positive control group, expression of ALP in echinacoside serum groups was obviously higher, there were significant differences(P<0.01). After using noggin, activity of ALP decreased, there were significant differences (P<0.05).10. Detection of expression of Smadl/5/8 during the process that different concentrations of echinacoside serum induced osteogenetic differentiation of BMSCs. Compared with the normal control group, expression of mRNA and protein of Smadl/5/8 in positive control group^ echinacoside group and Noggin which was inhibitor of BMP2 group increased obviously, there were significant difference (P<0.01). Compared with the positive control group, expression of mRNA and protein of Smadl/5/8 in echinacoside group increased obviously (P<0.01), and expression of mRNA、 Western blot and immunof luorescence staining of Smadl/5/8 in Noggin which was inhibitor of BMP2 group was obviously decreased, there were significant differences (P<0.01).11. Detection of expression of ZHX3 during the process that different concentrations of echinacoside serum induced osteogenetic differentiation of BMSCs. Compared with the normal control group, expression of mRNA and protein of ZHX3 in positive control group、 echinacoside group and Noggin which was inhibitor of BMP2 group increased obviously, there were significant difference (P<0.01). Compared with the positive control group, expression of mRNA and protein of ZHX3 in echinacoside group increased obviously (P<0.01), and expression of mRNA and protein of ZHX3 in Noggin which was inhibitor of BMP2 group was obviously decreased, there were significant differences (P<0.01).12. Detection of expression of Runx2 during the process that different concentrations of echinacoside serum induced osteogenetic differentiation of BMSCs. Compared with the normal control group, expression of mRNA and protein of Runx2 in positive control group、 echinacoside group and Noggin which was inhibitor of BMP2 group increased obviously, there were significant difference (P<0.01). Compared with the positive control group, expression of mRNA and protein of Runx2 in echinacoside group increased obviously (P<0.01), and expression of mRNA and protein of Runx2 in Noggin which was inhibitor of BMP2 group was obviously decreased, there were significant differences (P<0.01).13. Detection of expression of OSX during the process that different concentrations of echinacoside serum induced osteogenetic differentiation of BMSCs. Compared with the normal control group, expression of mRNA and protein of OSX in positive control group、 echinacoside group and Noggin which was inhibitor of BMP2 group increased obviously, there were significant difference (P<0.01). Compared with the positive control group, expression of mRNA and protein of OSX in echinacoside group increased obviously (P<0.01), and expression of mRNA and protein of OSX in Noggin which was inhibitor of BMP2 group was obviously decreased, there were significant differences (P<0.01).Conclusion:1. Within the certain concentration range of echinacoside serum (5%、7.5%、 10%、15% medicated serum) could promote proliferation of BMSCs with a concentration dependent manner. Low concentrations (2.5%) or high concentration (20%) had no effect on proliferation of BMSCs.2. Echinacoside serum could make the expression of ALP, BGP, collagen I on BMSCs increase, promote the formation of mineralized nodules, those of result prompted, echinacoside serum had the ability of inducing BMSCs to the osteoblast differentiation, of which 10% echinacoside serum was the best results.3. Echinacoside could promote expression of mRNA and protein of BMP2, Smadl/5/8, ZHX:3, Runx2 and OSX in BMSCs significantly, the mechanism was possibly related to BMP2-Smad-Runx2 pathway.