| Objective: 1.When TNBS/ethanol induced the inflammatory response in rat UC model,whether the bone marrow source BMSCs was mobilized into the peripheral blood,the BMSCs from the peripheral blood and the BMSCs from the bone marrow,the biological characteristics of the proliferation and migration adherence were different,which lay the foundation for the study of the BMSCs homing repair of the colonic mucosa.2.To explore the different intervention effects of Shenling Baizhu powder and Tongxie Yaofang decoction on the mobilization of BMSCs in TNBS/ ethanol induced rats UC model,the growth and proliferation,the migration and adhesion of BMSCs from the source of peripheral blood and the bone marrow source BMSCs.At the same time,guided by the theory of "syndrome correlation",we evaluated the TCM syndrome characteristics of TNBS/ induced UC rat model through two different prescription intervention effects.Method: 1.180-220 g health SPF 20 SD male rats,conventional breeding after 1 week or so,a random sample 6 as blank group,all the rest were built UC model.Selected by the method of the trinitrobenzene sulfonic acid(TNBS)/ 50% ethanol to build 5 weeks time,end of the building,in colon tissue,using HE staining to successful evaluation model.2.According to the original composition and dosage ratio of Shenling Baizhu powder and Tongxie Yaofang decoction,using traditional Chinese medicine decoction methods to fill the stomach medicine,randomly selected 2 group from the model group were given henling Baizhu powder and Tongxie Yaofang decoction treatment,the other group was given the same dose of normal saline lavage,after 28 days,anesthesia in rats,collecting the blood,separating the femur,collecting blank group serum and Shenling Baizhu powder and Tongxie Yaofang decoction medicated serum.Extract each group of peripheral blood and bone marrow BMSCs to cultivation.Using inverted microscope to observe the form of the BMSCs,the immunofluorescence to test the BMSCs of peripheral blood and bone marrow,the flow cytometry technology to detect the bone marrow and peripheral blood mononuclear cells.3.Taking 20 weight for 180-220 g of SPF healthy SD male rats,after 1 week of the conventional adaptive breeding,random in 2 groups,each group have 10,fasting 12 to 24 h before experiment,each group rat with Shenling Baizhu powder and Tongxie Yaofang decoction to fill the stomach,blood time: on the treatment of third time,after 2h,aseptic conditions,abdominal aortic blood sampling,then let stand,centrifugal,the separation of serum,mix the drug serum,water bath inactivated,storage of refrigerator at-20 ?.4.Taking groups of peripheral blood and bone marrow BMSCs with 0.25% of trypsin digestion,according to 1:2 go down to posterity,the third generation of BMSCs as a follow-up experiment,using flow cytometry technique to detect the growth of each source of BMSCs,using RTCA testing the adhesion ability and the migration of BMSCs in each group and observing the intervention of the Shenling Baizhu powder and Tongxie Yaofang decoction drug-containing serum,using the source of immunohistochemical method and the source of QPCR to detect peripheral blood groups of the expression of VCAM 1,using the flow cytometry technique to detect the expression of peripheral blood VLA-4.Results: 1.The mobilization detection of BMSCs in bone marrow and peripheral blood source.The percentage of positive cells in the bone marrow source of model group was shown decreased compared with the blank group,the difference was statistically significant(P<0.05).Compared with the model group,the percentage of positive cells in the group of Shenling Baizhu group and the Tongxie Yaofang group was increased,and the performance of Tongxie Yaofang group was more obvious,and the difference was statistically significant(P<0.05).The percentage of positive cells from the peripheral blood source of model group was shown increased compared with the blank group,the difference was statistically significant(P<0.05).Compared with the model group,the percentage of positive cells in the group of Shenling Baizhu group and the Tongxie Yaofang group was decreased,and the performance of Tongxie Yaofang was more obvious,and the difference was statistically significant(P<0.05).2.The morphological observation of BMSCs by inverted microscope.The form of the vaccination cells mainly for the shape of oval and round,the cells densely suspended in the culture medium,the refractive character is stronger,in three days later,some cells shape present class round or spindle,there are still a large number of adherent cells suspension in the medium,the first generation of cells is shown that the cells present adherent growth,the shape present spindle,irregular and a variety of forms,such as triangle or polygon,the fusion in the range of 80-90%,and the mixed cells suspended is less in the culture medium.3.The cycle of BMSCs in bone marrow and peripheral blood source was detected by RTCA.The cycle of BMSCs in each group of the bone marrow source presented that the proportion of S phase cells in the model group shown increased compared with blank group,and the difference was statistically significant(P<0.05).The proportion of s-phase cells in the group of Shenling Baizhu group and the Tongxie Yaofang group shown increased compared with model group,and the increase of the Tongxie Yaofang group was more obvious,and the difference was statistically significant(P<0.05).The cycle of BMSCs cells in each group of peripheral blood was presented that the proportion of S phase cells in the model group shown increased compared with the blank group,and the difference was statistically significant(P<0.05).The proportion of s-phase cells in the group of Shenling Baizhu group and the Tongxie Yaofang group shown increased Compared with the model group,and the increase of the Tongxie Yaofang group was more obvious,and the difference was statistically significant(P<0.05).4.The proliferation ability of BMSCs originated from bone marrow and peripheral blood source was detected by RTCA.BMSCs in each group of bone marrow,compared with the blank group,the cell activity of the model group increased(P<0.05)and the difference was statistically significant;Compared with the model group,the cell activity of the group of Shenling Baizhu powder and Tongxie Yaofang Group was higher than that of the model group(P<0.05),the difference was statistically significant;and the cells of the Shenling Baizhu powder and the Tongxie Yaofang group were significantly different from that of the model group.The activity was similar(P>0.05).BMSCs in each group of peripheral blood,the cell activity of the model group was lower than that in the blank group(P<0.05),and the cell activity of the model group was the lowest in the four groups.The cell activity of the group of Shenling Baizhu powder and Tongxie Yaofang Group was higher than that of the model group,the cell activity of the Tongxie Yaofang group was the highest(P<0.05),and the difference had the combination of the model group(P<0.05),the difference was statistically significant.5.The adhesion properties of BMSCs originated from peripheral blood source was detected by RTCA.Compared with the blank group,the cell index of the model group was close to 0(P>0.05).Compared with the model group,with the time flying,the cells attached more and more in the group of Tongxie Yaofang group(P<0.05),and the difference was statistically significant,but the relationship with the group of Shenling Baizhu powder group was weaker(P>0.05).6.To detect the expression of VCAM-1 in each group of peripheral blood sources by QPCR and immunohistochemistry.The expression of VCAM-1 in the model group was increased compared with the blank group,and the difference was statistically significant(P<0.05),Compared with the model group,the expression of VCAM-1 in the group of Shenling Baizhu group and the Tongxie Yaofang group was increased,and the expression of VCAM-1 in the Tongxie Yaofang group was significantly higher,and the difference was statistically significant(P<0.05).The immunohistochemical staining showed that the expression of VCAM-1 was a brown irregular shape,which could be detected in all groups.7.The expression of VLA-4 in peripheral blood was detected by the flow cytometry.The expression of VLA-4 in peripheral blood source presented that the expression of VLA-4 in the model group was increased compared with the blank group,and the difference was statistically significant(P<0.05).Compared with the model group,the expression of VLA-4 in the group of Shenling Baizhu group and the Tongxie Yaofang group were increased,and the expression of VLA-4 in the Tongxie Yaofang group was more obvious,and the difference was statistically significant(P<0.05).8.RTCA was used to detect the migration of BMSCs in peripheral blood source and the difference migration ability on the drug containing serum.BMSCs in each group of peripheral blood,compared with the blank group,the CI increased in the model group(P<0.05)and the difference was statistically significant.Compared with the model group,the CI in the group of Shenling Baizhu powder and the Tongxie Yaofang group increased,and the increase of CI in the Tongxie Yaofang Group was higher than that of the Shenling Baizhu powder group(P<0.05),the difference was statistically significant.Conclusion: 1.In the process of ulcerative colitis,which has the function of motivating the bone marrow-derived BMSCs to the peripheral blood,the Shenling Baizhu powder and the Tongxie Yaofang decoction have the function of promoting the bone marrow-derived BMSCs increase and the peripheral blood source of BMSCs reduction,the group of Tongxie Yaofang decoction is better than the group of Shenling Baizhu powder.2.It can accelerate the growth of the bone marrow and the peripheral blood sources of BMSCs in the process of UC,and the Shenling Baizhu powder and the Tongxie Yaofang decoction can accelerate the growth of the BMSCs in bone marrow and peripheral blood sources,and the Tongxie Yaofang group showed more obvious.3.It can promote the proliferation of the bone marrow originated of BMSCs in each group in the process of UC,the Shenling Baizhu powder and the Tongxie Yaofang decoction has the function of promoting proliferation of the bone marrow originated of BMSCs,they function of promoting proliferation was comparatively.The BMSCs of the peripheral blood proliferation effect is suppressed in the process of UC,the Shenling Baizhu powder and the Tongxie Yaofang decoction has the function of promoting proliferation of BMSCs of peripheral blood,and the Tongxie Yaofang decoction was more obvious.4.In the process of UC,the adhesion ability of BMSCs will be increased by the serum containing medicine concentration of Tongxie Yaofang decoction in the peripheral blood source,and its concentration was positive hours-dependent,the relationship with Shenling Baizhu powder was not significant.In addition,Shenling Baizhu powder and Tongxie Yaofang decoction have the function of promoting the migration,and Tongxie Yaofang decoction was more obvious.5.Shenling Baizhu powder and the Tongxie Yaofang decoction have the function of promoting the expression of UC rats VCAM-1 and the VLA-4 in each group of peripheral blood source BMSCs,the Tongxie Yaofang decoction was more apparent.The adhesion characteristic of BMSCs has the relationship with the higher expression of VCAM-1 and VLA-4. |
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