| ObjectivesTo observe the influence of Bushen Tuihuang (BSTH) Decoction medicated serum from severe chronic hepatitis or chronic severe hepatitis patients on the rat marrow stem cells (MSCs) proliferation, discussing a novel understanding to the pathology of "critical disease harms kidney", as well as the mechanisms of BSTH Decoction treating the above hepatitis, so as to elucidate the biological basis of the "kidney deficiency induced jaundice" syndrome, and provide an experimental basis in enhancement of clinical curative effect.MethodsThe type B viral hepatitis patients who were accorded with the diagnosis criteria of severe chronic hepatitis or the chronic severe hepatitis, and the TCM diagnosis criteria of "kidney deficiency induced jaundice" syndrome, were divided into 2 "groups randomly and treated respectively with western medicine and western medicine plus Chinese medicine functions in tonifying kidney and removing jaundice. Clinical indexes and laboratory index before and after the treatment were recorded and patients' serum was collected for observation.Methods of density gradient centrifugation and wall sticking were utilized to separate and purify the rat MSCs. Keep the serum of chronic heavy hepatitis patients who used it in the artificial liver ("artificial liver serum" for short). The artificial liver serum was added to MSCs culture medium and the research was conducted in two groups as non-FBS group and 10% FBS contained group, they each were cultured with MSCs with artificial liver serum of different concentrations. MTT assay was used for determination of OD value of each MSCs group, which reflected the proliferation of cells, and then determined a proper concentration of medicated serum for MSCs proliferation.Rat MSCs were cultured with serum from patients with "kidney deficiency induced jaundice" syndrome. The experiment was conducted in 4 groups: Group A ( normal serum for control); Group B (serum from patients upon hospitalization); Group C (received western medicine treatment): Group D (western medicine plus tonifying kidney and removing jaundice treatment). The serum was used for culturing rat MSCs for 8 days, cells growth were observed every day, and meantime MTT assay was applied for attaining OD value indicating MSCs growth and the growth curve was drown according to mean of OD value.All data were analyzed with SPSS 12.0 software. Measurement data were expressed as mean±SD, t-test was used. ResultsThe symptom scores of western medicine group and western medicine plus BSTH group showed no significant difference (P>0.05) before treatment, and after treatment, the western medicine plus BSTH group showed obvious improvement, the symptom score (8.37+1.99) was lower than western medicine group (10.13±2.47), statistical analysis showed significant difference (P<0.05). Both groups demonstrated TBiL decline after treatment and were of significant differences comparing with that before treatment (for western medicine 300.33±45.04; western medicine plus BSTH group 322.35±72.26) (P<0.05), whereas no significant difference was observed between the two groups after treatment (P>0.05).The MSCs growth was inhibited by the L-DMEM with respectively 15%, 17.5%, 20% artificial liver serum and no FBS. (P<0.05), and the artificial liver serum of 7%, 8%, 9% concentration with 10% FBS contained L-DMEM have certain promoting effect on MSCs growth (P<0.05).MSCs developed as cell colony about 4 days after treatment, and about 6 days there were polygon cell surroundings the cell colony; at about 7-8 days bigger colonies were formed and located in the center were undifferentiated cells, the surrounding cells were polygon cells, arrayed radially.OD values of the four groups varied with the time. The trend line generally began with upward, after raising to the plateau phase it kept stability or slight drop. OD value trend of each group at each time phases were similar, only with extent: Control group got into logarithms growth period earliest, its OD value obvious elevated, and at the 6 days it was close to stability; Western medicine plus BSTH treatment group and the western medicine group turned into a logarithms growth period at the 3-4 days, with that the western medicine plus BSTH treatment group had a more obvious elevation. Hospitalized group was not as obviously as rest of the other 3 groups, and get into plateau phase at the 6th day. The 3-7 days western medicine plus BSTH treatment group was significantly different from that western medicine group on OD value (P<0.01, P<0.05).ConclusionsSevere chronic hepatitis or the chronic severe hepatitis may manifest "kidney deficiency induced jaundice" in the pathological process, method of "tonifying kidney and removing jaundice" enhances the clinical curative effect.In the progress of the Chronic liver disease, besides the knowledge of "chronic disease harms the kidney", there is also a new understanding on the pathology as "severe disease harms the kidney". Their common biological basis may be closely related to with liver rebirth mechanisms as MSCs transforming into liver cells.Culturing cells with serum of patient with chronic liver disease is feasible and novel experimental approach. BSTH medicated serum is beneficial for MSCs proliferation. Combination research of culturing cells with serum from patient with chronic liver disease and the MSCs proliferation, transformation is a new way of research and a novel method for studying the essence of the integrated disease (chronic liver diseases) and syndrome (Yin deficiency in liver and kidney, deficiency of kidney induced jaundice, etc.). |
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