CN104962515A - Application of rhizome panacis majoris saponin inducing stem cells differentiating hepatic cells and hepatosis curing medicine - Google Patents
Application of rhizome panacis majoris saponin inducing stem cells differentiating hepatic cells and hepatosis curing medicine Download PDFInfo
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Abstract
The invention discloses application of rhizome panacis majoris saponin inducing stem cells differentiating hepatic cells and hepatosis curing medicine and belongs to the technical field of medicine. According to the application of rhizome panacis majoris saponin inducing stem cells differentiating the hepatic cells and the hepatosis curing medicine, rhizome panacis majoris saponin is used as the main component of an inducing solution to induce mesenchymal stem cells to directionally differentiate into hepatic related cells in vitro. According to the application of rhizome panacis majoris saponin inducing stem cells differentiating the hepatic cells and the hepatosis curing medicine, the new application of the rhizome panacis majoris saponin is developed, namely, the bone marrow stem cells are induced to directionally differentiate into hepatic related cells in vitro, the rhizome panacis majoris saponin is used for developing medicine for treating hepatosis and the moderate and advanced liver cancer, the pharmacology and the efficacy of the rhizome panacis majoris saponin are further defined and developed, the rhizome panacis majoris saponin is used as medicine raw materials to be put into medicine for curing the hepatosis, basis is provided for new medicine development, and the invention further provides an implementation method using rhizome panacis majoris saponin inducing the mesenchymal stem cells directionally differentiating hepatic cells.
Description
Technical field
The present invention relates to medical art, be specifically related to the application of Rhizome of Bipinnatifid Ginseng saponin(e differentiation of stem cells liver cell and treatment liver disease drug.
Background technology
China due to hepatitis b virus infection ratio higher, the infected so suffer from acute, chronic hepatitis, hepatic fibrosis and even liver cirrhosis, liver cancer event of common occurrence, hepatic diseases is one of main burden having become Health Care in China at present.When hepatic diseases proceeds to the End-stage liver disease such as liver cirrhosis (end-stage liver disease, ESLD), usually involve extensive hepatic parenchymal cells, hepatic tissue regenerative power declines, and causes liver function lose compensatory and finally cause liver failure.Conventional internal medicine liver protecting therapy poor effect, case fatality rate is very high.At present, patients with orthotopic liver transplantation is the most effective means for the treatment of ESLD, although short term effect is better, because complexity of performing the operation, organ origin are difficult, costly, the factor such as immunological rejection and life-time service immunosuppressor, its wide clinical application is made to receive certain restriction.The treatment End-stage liver disease that develops into of stem cells technology brings new hope in recent years.Stem cell therapy is simple because of it, and wound is little and become the focus of concern.Bone Marrow Stem Cells Transplantation has had very large progress in treatment acute and chronic liver failure, End-stage liver disease and hereditary metabolic disorders hepatopathy etc., it obtains certainly in short term effect, security, tolerance etc., and more and more comes into one's own because of the advantage (as low cost, convenience of drawing materials, there is not immunological rejection etc.) of itself.Although liver transplantation is the effective ways for the treatment of various End-stage liver disease, due to many for liver source deficiency, complicated operation, complication, transplant after the reason such as immunological rejection and medical expense costliness, limit the development of Clinical Liver Transplantation.Therefore, since carrying out stem-cell research, bone marrow stem cell (bone marrow stem cells, BMSCs) is transplanted as ESLD patient brings Gospel.
Stem cell is the initiating cell that a class has self and multi-lineage potential.Embryonic stem cell and BMSCs can be divided into according to its living stage.Embryonic stem cell has totipotency, histocytes all in body can be divided into, but, the directed differentiation of stem cell completes under the acting in conjunction of intraor extracellular factor, interaction between cell-ECM and some somatomedin can impel stem cell to break up to a direction, therefore, in microenvironment complicated in vivo, be difficult to guarantee that it breaks up to single direction, research finds the risk having formation tumour (as teratoma) after this myeloid-lymphoid stem cell is transplanted, simultaneously again because ethics problem, limit its development and application clinically.Many scientists successively confirm that the differentiation capability of BMSCs is the adult stem cell closest to embryonic stem cell, can break up multiple somatocyte such as becoming the heart, liver, brain, muscle, cartilage, pancreas islet in recent years.BMSCs is because of its abundance, and autotransplantation can keep away immunological rejection, does not relate to the plurality of advantages such as ethics and receives much attention, fully developing BMSCs, the treatment for vitals fatal disease such as the heart, liver, brains is opened up a new road.The people such as Jiang study and find the orientable hepatic lineage that is induced to differentiate into of BMSCs, and this cell has the morphology and function feature of normal liver cell, can synthesize albumin, alpha-fetoprotein, urea etc.Although also have liver stem cells, navel blood stem cell etc. to be applied to the report of clinical treatment ESLD.China also just carries out Chinese medicine as far back as 20 middle of century and studies the biological impact of BMSCs, and application treatment by Chinese herbs hemopathy, anti-chemicotherapy side effect etc. achieve good clinical effectiveness.
Rhizome of Bipinnatifid Ginseng (Panax japlcus var, major) is araliaceae ginseng plant.Another name Pestalotia funera, button seven, Rhizoma Panacis Japonici, pimple seven, Herba Senecionis Chrysanthemoidis, dish seven, wild pseudo-ginseng etc.Its dry root stock of commonly using among the people is used as medicine.Its bitter, sweet, be slightly cold, return liver, lung, stomach warp, there is the effects such as tonifying lung yin-nourishing, stasis-dispelling and pain-killing, hemostasis, cure mainly that deficiency of both qi and yin, dysphoria with smothery sensation are thirsty, cough due to consumptive disease, arthralgia, hemoptysis, haematemesis, bleeding from five sense organs or subcutaneous tissue, uterine bleeding and traumatic hemorrhage etc.Its chemical constitution study is shown, its main chemical compositions is saponins, be divided into oleanane type, dammarane type, steroid class, comprise Rhizome of Bipinnatifid Ginseng saponin(e K1 and K2, rhizome of Japanese Ginseng glycosides, 20-O-glucosyl group-ginsenoside Rf, ginsenoside Ro, ginsenoside Kd, Re, Rc, Rb, Rg1, Rb3, Rg2, GF1 ~ F3,20 (S)-panicled fameflower root saponin(e F11, Rhizome of Bipinnatifid Ginseng saponin(e F1 ~ F6, Rhizoma Panacis bipinnatifidi glycosides, notoginsenoside Sap-3 are β-sitosterol-3-O-β-D-glucopyranoside.Oleanolic Acid-28-0-β-D-adjoin feed glucoside, Oleanolic Acid, β-sitosterol-3-0-D-adjoin glucopyranoside glycosides, β-sitosterol etc.; Secondly be polysaccharide, sesquiterpenoids and micro elements needed by human Fe, Cu, Zn, Mn, Ni, Co, Mo etc.
Rhizome of Bipinnatifid Ginseng and main component Rhizome of Bipinnatifid Ginseng total saponins thereof have pharmacological action more widely, all have effect to blood and hemopoietic system, Digestive tract, immunity system, tumour, painstaking effort brain guard system etc.We study discovery, Rhizome of Bipinnatifid Ginseng has more significant anti-tumor activity, confirm that Rhizome of Bipinnatifid Ginseng can help body healthy tendency, recover or improve the stable state of organismic internal environment, forward regulates NK-IL-2 s function, and is divided into mature T cells by short thymocyte hyperplasia and discharges to periphery, raising Organism immunoregulation factor level further, promote the activity of anti-tumour effect cell, to remove or the developing of Tumor suppression; Human hepatoma cell strain SMMC27721 can be brought out and produce typical apoptosis form and Change of Ultrastructure; Cell block can be made in the G0/G1 phase, thus stop cell to be changed to the S phase, interference cell cycle progression, and significantly cause apoptosis, reduce oncogene c-myc and c-fos and express, increase Suppressor p53 and p21 expression.
At present, though there is the report material about differentiation of stem cells hepatoblast, but have not been reported for the research of application aspect of Rhizome of Bipinnatifid Ginseng saponin(e differentiation of stem cells liver cell and treatment hepatopathy.Rhizome of Bipinnatifid Ginseng has good curative effect to liver injury, and confirm that Rhizome of Bipinnatifid Ginseng saponin(e can induce BMSCs to be divided into liver cell, but how relevant Rhizome of Bipinnatifid Ginseng saponin(e mobilizes BMSCs to raise to damaged liver, go back to the nest at present, how to improve pathology liver cell microenvironment, promotion is raised the survival of BMSCs and is divided into hepatic lineage, thus reparation damaged liver, recover the Mechanism Study also rare report of damaged liver function; China is hepatopathy big country, and liver cirrhosis, liver cancer and liver failure have become the final home to return to of numerous ESLD patient.Stem cell transplantation is that treatment End-stage liver disease provides new hope, utilize the hepatocellular research of differentiation of stem cells also just to become present stage problem demanding prompt solution, and the medicine for the treatment of hepatopathy aspect also has market widely.
Summary of the invention
One of main purpose of the present invention is to meet the need of market, solve differentiation of stem cells liver cell difficulty to realize and hepatopathy problem difficult to treat, thus provide the novelty teabag of Rhizome of Bipinnatifid Ginseng saponin(e, i.e. the application of Rhizome of Bipinnatifid Ginseng saponin(e differentiation of stem cells liver cell and treatment liver disease drug.
This invention exploits the novelty teabag of Rhizome of Bipinnatifid Ginseng saponin(e, comprising: utilize Rhizome of Bipinnatifid Ginseng saponin(e inducing bone mesenchymal stem cell directional to be divided into liver cell; Rhizome of Bipinnatifid Ginseng saponin(e is utilized to develop the medicine for the treatment of hepatopathy and mid and late liver cancer.Present invention also offers and utilize Rhizome of Bipinnatifid Ginseng saponin(e inducing bone mesenchymal stem cell directional to be divided into hepatocellular implementation method.
For solving the problems of the technologies described above, technical scheme provided by the invention is:
The hepatocellular application of Rhizome of Bipinnatifid Ginseng saponin(e differentiation of stem cells, the Rhizome of Bipinnatifid Ginseng saponin(e extracted from Rhizome of Bipinnatifid Ginseng is application in liver cell inductor preparing differentiation of stem cells.
Further, the hepatocellular application of Rhizome of Bipinnatifid Ginseng saponin(e differentiation of stem cells, described stem cell is adult stem cell.
Further, the hepatocellular application of Rhizome of Bipinnatifid Ginseng saponin(e differentiation of stem cells, described adult stem cell is mesenchymal stem cells MSCs.
Further, the hepatocellular application of Rhizome of Bipinnatifid Ginseng saponin(e differentiation of stem cells, described differentiation of stem cells is the induced liquid that the main component of liver cell induced liquid comprises Rhizome of Bipinnatifid Ginseng saponin(e, pHGF (HGF), the differentiation of osteocyte direction.
The hepatocellular application method of a kind of Rhizome of Bipinnatifid Ginseng saponin(e differentiation of stem cells: get rat bone marrow mesenchymal stem cells and carry out Secondary Culture, get the stable mesenchymal stem cells MSCs gone down to posterity and carry out inducing culture in inducing culture, obtain ripe hepatocyte-like cells; Described inducing culture is the basic medium being added with Rhizome of Bipinnatifid Ginseng saponin(e and cell growth factor, in described basic medium, the addition of Rhizome of Bipinnatifid Ginseng saponin(e and cell growth factor is: the concentration of Rhizome of Bipinnatifid Ginseng saponin(e is: 50-200 μ g/mL, and the concentration of cell growth factor (HGF) is: 10-30ng/mL.
Preferably, the concentration of the thin HGF added in described basic medium is: 20ng/mL.
Further, the hepatocellular application method of Rhizome of Bipinnatifid Ginseng saponin(e differentiation of stem cells, get rat bone marrow mesenchymal stem cells and carry out Secondary Culture, get the mesenchymal stem cells MSCs of the third generation, be inoculated in low sugar DMEM substratum, culture condition is:, after described mesenchymal stem cells MSCs individual layer grows to 80%, the substratum of described mesenchymal stem cells MSCs is replaced by the inducing culture containing Rhizome of Bipinnatifid Ginseng saponin(e and cell growth factor and carries out inducing culture, within every 3 days, change liquid 1 time, after cultivating 2 weeks, obtain ripe hepatocyte-like cells.
The hepatocellular inductor of a kind of differentiation of stem cells, described inductor contains Rhizome of Bipinnatifid Ginseng or Rhizome of Bipinnatifid Ginseng saponin(e.
Rhizome of Bipinnatifid Ginseng saponin(e treatment hepatopathy in an application, described in be applied as Rhizome of Bipinnatifid Ginseng saponin(e treatment hepatopathy in pharmaceutical composition in application.
Treat the pharmaceutical composition in hepatopathy, containing Rhizome of Bipinnatifid Ginseng saponin(e in the pharmaceutical composition in described treatment hepatopathy; The dosage form of described pharmaceutical composition comprises: injection, tablet, capsule, aerosol, suppository, film, pill, ointment, control-released agent, sustained release dosage or nanometer formulation.
Beneficial effect of the present invention is: the novelty teabag developing Rhizome of Bipinnatifid Ginseng saponin(e: utilize Rhizome of Bipinnatifid Ginseng saponin(e inducing bone mesenchymal stem cell in vitro directed differentiation to be liver system cell, and differentiation is stable, effective; Be applied to by Rhizome of Bipinnatifid Ginseng saponin(e in the medicine for the treatment of hepatopathy, the pharmacology of further distinct Rhizome of Bipinnatifid Ginseng saponin(e and drug effect, for the exploitation of new drug provides the foundation.
Accompanying drawing explanation
Figure 1 shows that mesenchymal stem cells MSCs (BMSCs) cellular form figure: A: BMSCs cellular form figure, B before dyeing: BMSCs cellular form figure after dyeing;
Fig. 2 is BMSCs cell growth curve;
Cellular form figure: A: blank group, B: Rhizome of Bipinnatifid Ginseng saponin(e 200 μ g/mL group after Fig. 3 Rhizome of Bipinnatifid Ginseng saponin(e induction BMSCs differentiation;
The effect diagram of cell glycogen biosynthesis after Fig. 4 Rhizome of Bipinnatifid Ginseng saponin(e induction BMSCs differentiation: A: blank group, B: Rhizome of Bipinnatifid Ginseng saponin(e 200 μ g/mL group;
Fig. 5 Rhizome of Bipinnatifid Ginseng saponin(e induction BMSCs breaks up the effect diagram of cell AFP, Albumin, CK18 protein expression after 7 days: 1: blank group, 2:HGF group, 3: Rhizome of Bipinnatifid Ginseng saponin(e 50 μ g/mL group, 4: Rhizome of Bipinnatifid Ginseng saponin(e 100 μ g/mL group, 5: Rhizome of Bipinnatifid Ginseng saponin(e 200 μ g/mL group;
Fig. 6 Rhizome of Bipinnatifid Ginseng saponin(e induction BMSCs breaks up the effect diagram of cell AFP, Albumin, CK18 protein expression after 14 days: 1: blank group, 2:HGF group, 3: Rhizome of Bipinnatifid Ginseng saponin(e 50 μ g/mL group, 4: Rhizome of Bipinnatifid Ginseng saponin(e 100 μ g/mL group, 5: Rhizome of Bipinnatifid Ginseng saponin(e 200 μ g/mL group;
The effect diagram that Fig. 7 Rhizome of Bipinnatifid Ginseng saponin(e is raised in Liver Fibrosis Model liver tissues of rats BMSCs;
Fig. 8 Rhizome of Bipinnatifid Ginseng saponin(e and BMSCs transplant HE dyeing effect diagram: the A to Liver Fibrosis Model rat liver fibrosis: normal group, B: model group, C:BMSCs transplantation group, D: Rhizome of Bipinnatifid Ginseng saponin(e group, E:BMSCs transplanting+Rhizome of Bipinnatifid Ginseng saponin(e group;
Fig. 9 Rhizome of Bipinnatifid Ginseng saponin(e and BMSCs transplant the impact on Liver Fibrosis Model liver tissues of rats Nrf2 protein expression: 1: normal group, 2: model group, 3:BMSCs transplantation group, 4: Rhizome of Bipinnatifid Ginseng saponin(e group, 5:BMSCs transplanting+Rhizome of Bipinnatifid Ginseng saponin(e group.
Embodiment
Hereafter will describe embodiments of the invention in detail by reference to the accompanying drawings.It should be noted that the combination of technical characteristic or the technical characteristic described in following embodiment should not be considered to isolated, they can mutually be combined thus be reached better technique effect.
The separation of embodiment 1 mesenchymal stem cells MSCs (BMSCs), cultivation and qualification
Test animal is: male Sprague-Dawley (SD) rat, quality (200 ± 20) g, is provided by Wuhan University's Experimental Animal Center, credit number: SCXK (Hubei Province) 2008-0004, sub-cage rearing, freely drinks water and ingests.
Aseptically put to death SD rat, take out femur fast, repeatedly rinse medullary space with the cell culture fluid containing foetal calf serum 10%, collect bone marrow cell suspension, piping and druming mixing, centrifugal going contains heteroproteose cell and erythrocyte splitting fragment supernatant, collecting precipitation, is suspended in the NH of precooling fast
4splitting erythrocyte 8min in the hypotonic salts solution of Cl, centrifugally removes supernatant, and the cell of collecting precipitation, counts, and adjustment cell density to 2 × 106/L, is inoculated in Tissue Culture Dish, is placed in CO
2cultivate in incubator, change liquid cell after 48h, when cell monolayer grows to 80%, carry out Secondary Culture.
1, the qualification of Cell viability
Cell is prepared into single cell suspension, gets 100 μ L cell suspensions, add equivalent 0.5% and expect blue dye liquor, mix fast, dye, carry out cell counting with tally, calculate Cell viability.
As shown in Figure 1, basis of microscopic observation visible cell is that spindle shape, size are even, and transparence is good; Growth of Cells is relatively slow in early days, breeds rapidly afterwards, 3 days visible obviously Colony forming afterwards.
2, passage and growth curve measure
When cell monolayer grows to 80%, draw supernatant, add 0.1% collagenase IV liquid 1mL and digest 3min, then add nutrient solution and stop digestion, the centrifugal 2min of 1500rpm, with nutrient solution Eddy diffusion cell, adjustment cell density to 2 × 104/L is inoculated in culture dish and cultivates.The cell got after 3 generations of going down to posterity carries out cell growth curve mensuration.Collect the normal cell of logarithmic phase and tumour cell, adjustment concentration of cell suspension and with 1 × 105 cell concn kind 96 orifice plate, every hole adds 100 μ L, and surrounding PBS seals, in 5%CO
2, hatch 12h, 5%CO for 37 DEG C
2, cultivate 24 hours, then add MTT (5mg/mL) 20 μ L for 37 DEG C, continue cultivation and stop after 4 hours cultivating, take out 96 orifice plates, carefully remove liquid in hole, every hole adds 150 μ LDMSO, and concussion shakes up 10min, and crystallisate is fully dissolved.Survey its OD value under microplate reader 490nm wavelength, and calculate inhibiting rate.
Calculation formula: inhibiting rate=(1-medicine hole OD/ blank well OD) × 100%.
Fig. 2 is BMSCs cell growth curve, and as shown in Figure 2, BMSCs is when cultivating 1-2 days, and cell number has and to a certain degree reduces, and after 3 days, cell increases sharply, and carries out growth logarithmic phase; Cultivate after 8 days, cell carries out growth climax, and the speed of growth reduces gradually afterwards, enters the growth platform phase.
3, the morphological observation of cell
Change liquid at every turn and observe under inverted microscope respectively before going down to posterity and take pictures, record cellular form.
4, the detection of the surface antigen of cell
Regular cell, the centrifugal 5min of 800rpm, abandon supernatant, the cell of collection is placed in the PBS liquid of precooling, repeats 3 times, to remove cell debris, adjustment cell density to 1 × 108/ is managed, then add fluorescently-labeled antibody (FITC-CD44, FITC-CD90, PE-CD45) mark respectively, after PBS washing, use flow cytomery.
Flow cytomery result shows, and from rat femur, BMSCs is separated surface antigen CD44, CD90 positive expression of the cell obtained, and CD45 does not express.
5, statistical study
Every test at least repeats 3 times, and the data of all experimental results are with mean ± standard deviation
represent, data analysis is carried out on SPSS13.0 statistical package, the comparison of many group differences is carried out with one-way analysis of variance (one-way ANOVA), compare the difference of mean between two groups with Dunnett-t inspection, P<0.05 illustrates that difference has statistical significance.
Above-mentioned BMSCs separation purification method makes the separation of BMSCs, it is convenient to cultivate; By analyzing being separated the cell obtained: this cell can express CD44, CD90 surface antigen that BMSCs has, and does not express the CD45 surface antigen that hematopoietic cell has; Confirm that this cell can be divided into the abilities such as scleroblast through induction simultaneously.It can thus be appreciated that being separated the cell obtained is BMSCs.
The external evoked BMSCs of embodiment 2 Rhizome of Bipinnatifid Ginseng saponin(e is divided into hepatic lineage
Test animal is: male Sprague-Dawley (SD) rat, quality (200 ± 20) g, is provided by Wuhan University's Experimental Animal Center, credit number: SCXK (Hubei Province) 2008-0004, sub-cage rearing, freely drinks water and ingests.
Test medicinal material is: Rhizome of Bipinnatifid Ginseng medicinal material purchased from shenlongjia, through SanXia University's Natural products research with utilize key lab of Hubei Province Wang Lam to plant the root stock that professor is accredited as araliaceae ginseng plant's Rhizome of Bipinnatifid Ginseng.Rhizome of Bipinnatifid Ginseng saponin(e is by SanXia University's Natural products research and utilize key lab of Hubei Province to provide, and its Rhizome of Bipinnatifid Ginseng saponin content is 92.65%.
According in embodiment 1 describe method BMSCs is separated and Secondary Culture, get growth conditions good the 4th generation cell, routine digests, abundant cell dispersion, counting, the density of cell suspension is adjusted to 1 × 10
5individual/mL, be inoculated in 6 porocyte culture plates, every hole adds 2.5mL cell suspension; When cell complete fusion growth, discard substratum, wash cell 3 times with D-Hank ' s liquid, change inducing culture into, every 3 days replaced medium 1 time; 0-3 days, starts to carry out experiment on the 4th day and is grouped into: blank group, HGF group (15ng/mL), Rhizome of Bipinnatifid Ginseng saponin(e (50,100 and 200 μ g/mL) group and Rhizome of Bipinnatifid Ginseng saponin(e (100 μ g/mL)+HGF group (15ng/mL) coupling group.
1, morphological observation
Each group of BMSCs in 6 well culture plates is seeded in after respective handling, in its form of basis of microscopic observation by above-mentioned.
Visible under microscope: after 3 days, its original fusiformis form there occurs obvious change, there is circle, after 7 days its morphological specificity and liver cell similar, after 14 days, most BMSCs presents the circular feature of similar liver cell sample, and does not occur above-mentioned change by the blank group of HGF or the induction of Rhizome of Bipinnatifid Ginseng saponin(e, as shown in Figure 3, Fig. 3 is Rhizome of Bipinnatifid Ginseng saponin(e induction BMSCs differentiation rear cellular form figure: A is blank group; B is Rhizome of Bipinnatifid Ginseng saponin(e 200 μ g/mL group.
2, the former detection of endocellular sugar
BMSCs is inoculated in 6 well culture plates of prefabricated cell climbing sheet, induce 0,7 and 14 day, after hatching 1h with 1% amylase solution 25 DEG C, take out creep plate with 4% paraformaldehyde fix after, with Schiff ' s agent treated 30min, rinse 3 times with Potassium hydrogen sulfite, after haematoxylin redyeing, observe at microscope.
BMSCs is after inducing 14 days through Rhizome of Bipinnatifid Ginseng saponin(e as shown in Figure 4, and blank group does not have the ability of glycogen biosynthesis, and HGF or Rhizome of Bipinnatifid Ginseng saponin(e group differentiation-inducing after cell there is the ability of glycogen biosynthesis.
3, the ability of differentiation-inducing rear cellular uptake LDL is measured
Cell induction, after 0,7 and 14 days, washes cell 3 times with D-Hank ' s liquid, after then hatching 24h with 10 μ g/mLDil-Ac-LDL substratum 37 DEG C, by spectrophotofluorometer (excitation wavelength 549nm, emission wavelength 565nm) fluorescence intensity.
As shown in Table 1, BMSCs through HGF, Rhizome of Bipinnatifid Ginseng saponin(e separately and with HGF combined utilization induce stimulate break up after significantly can increase the ability of cellular uptake LDL after differentiation, and be extended for 14 days by 7 days along with the differentiation-inducing time, its expression amount increases further, wherein Rhizome of Bipinnatifid Ginseng saponin(e 200 μ g/mL group action effect and HGF similar.
Table 1 Rhizome of Bipinnatifid Ginseng saponin(e induction BMSCs differentiation within 7,14 days, absorb afterwards LDL ability impact (
n=4)
| Group | Dosage | 7 days | 14 days |
| Blank group | ---- | 28.23±2.47 | 29.17±2.78 |
| HGF group | 20ng/mL | 50.93±2.31** | 70.53±7.39** |
| Rhizome of Bipinnatifid Ginseng saponin(e | 50μg/mL | 30.07±3.17 | 45.97±6.65* |
| Rhizome of Bipinnatifid Ginseng saponin(e | 100μg/mL | 34.83±2.06* | 52.73±4.17** |
| Rhizome of Bipinnatifid Ginseng saponin(e | 200μg/mL | 41.17±3.46** | 64.97±4.84** |
| Rhizome of Bipinnatifid Ginseng saponin(e+HGF group | 100μg/mL+20ng/mL | 59.36±2.17** | 80.12±3.69** |
Compare with blank group, * P < 0.05, * * P < 0.01
4, the ability of differentiation-inducing rear emiocytosis urea is measured
Cell induction, after 0,7 and 14 days, is transformed into the NH of 6mM
4the induced liquid of Cl continues to cultivate 24h, and then the centrifugal 10min of 1500rpm, gets supernatant, measures the content of urea in supernatant liquor with automatic clinical chemistry analyzer.
As shown in Table 2, BMSCs through HGF, Rhizome of Bipinnatifid Ginseng saponin(e separately and with HGF combined utilization induce stimulate break up after significantly can increase the ability of emiocytosis urea after differentiation, and be extended for 14 days by 7 days along with the differentiation-inducing time, its expression amount increases further, wherein Rhizome of Bipinnatifid Ginseng saponin(e 200 μ g/mL group action effect and HGF similar.
Table 2 Rhizome of Bipinnatifid Ginseng saponin(e induction BMSCs differentiation within 7,14 days, secrete afterwards urea ability impact (
n=4)
| Group | Dosage | 7 days (μ g/ hole) | 14 days (μ g/ hole) |
| Blank group | ---- | 3.31±0.23 | 3.55±0.27 |
| HGF group | 20ng/mL | 7.48±0.43** | 8.05±0.17** |
| Rhizome of Bipinnatifid Ginseng saponin(e | 50μg/mL | 4.12±0.31* | 4.87±0.42** |
| Rhizome of Bipinnatifid Ginseng saponin(e | 100μg/mL | 5.37±0.78** | 6.53±0.37** |
| Rhizome of Bipinnatifid Ginseng saponin(e | 200μg/mL | 6.26±0.30** | 7.01±0.16** |
| Rhizome of Bipinnatifid Ginseng saponin(e+HGF group | 100μg/mL+15ng/mL | 8.27±0.56** | 9.16±0.48** |
Compare with blank group, * P < 0.05, * * P < 0.01
5, real-time quantitative PCR detects HGF, c-MET, AFP, Albumin, CK18 genetic expression of the rear cell of differentiation
Respectively at 0,7 and 14 day after induction, extract the cell total rna after differentiation with Trizol reagent, by specification operates.It is 1.8 ~ 2.0 that total serum IgE measures 260nm and 280nm place's Reinhoit Zahl (D260/D280) through ultraviolet spectrophotometer.Be cDNA by the RNA reverse transcription of extraction, then increase, add in the reaction tubes of RNase Free process
premix Ex TaqTM II (2X) 12.5 μ L, PCR Forward Primer (10 μMs) 1 μ L, PCR ReversePrimer (10 μMs) 1 μ L, DNA profiling 2 μ L, dH
2o (sterile purified water) 8.5 μ L, end reaction system is 25 μ l.Sample is put into real-time quantitative PCR amplification instrument to increase 40 times by 95 DEG C of denaturation 30s, 95 DEG C of sex change 5s, annealing 30s, 72 DEG C of extension 20s conditions.Utilize PCR instrument remittance abroad amplification curve, carry out interpretation of result.HGF, c-MET, AFP, Albumin, CK18 and GAPDH (Shanghai Sheng Gong biotechnology company limited synthesizes, and concrete primer sequence is in Table).
Table 3 real-time quantitative PCR primer sequence table:
| Gene Name | Upstream primer sequence | Downstream primer sequence |
| HGF | GCGAGGAGAAACGCAAACAG | CCACGACCAGGAACAATGAC |
| c-MET | GGACGCCGCAGAGCAGAC | GCCCCCGCCTTGAACTT |
| AFP | GCTTCTACCACTGTCTGGGATG | GTCTGGAGCGGTCTTCTTGC |
| Albulnin | CTGTAGTGGGTCCCTGGTGG | GGGTAGCCTGAGATGGTTGTG |
| CK18 | GCCCTGGACTCCAGCAACT | ACTTTGCCATCCACGACCTT |
| GAPDH | GCTGGGGCTCACCTGAAGG | GGATGACCTTGCCCACAGCC |
From table 4-5, BMSCs through HGF, Rhizome of Bipinnatifid Ginseng saponin(e separately and with HGF combined utilization induce stimulate break up after significantly can increase liver cell specificity HGF, c-MET, AFP, Albumin, CK18 genetic expression, and be extended for 14 days by 7 days along with the differentiation-inducing time, its expression amount increases further, wherein Rhizome of Bipinnatifid Ginseng saponin(e 200 μ g/mL group action effect and HGF similar.
Table 4 Rhizome of Bipinnatifid Ginseng saponin(e induction BMSCs break up cell HGF, c-MET, AFP, Albumin, CK18 genetic expression after 7 days impact (
n=4)
Compare with blank group, * P < 0.05, * * P < 0.01
Table 5 Rhizome of Bipinnatifid Ginseng saponin(e induction BMSCs break up cell HGF, c-MET, AFP, Albumin, CK18 genetic expression after 14 days impact (
n=4)
Compare with blank group, * P < 0.05, * * P < 0.01
6, Western blot detects AFP, Albumin, CK18 protein expression of the rear cell of differentiation
Respectively at 0,7 and 14 day after induction, illustrate according to Protein Extraction Reagent kit, extract the albumen of the rear cell of different treatment group differentiation, after carrying out protein quantification, add sample buffer and boil 5min, respectively get 20 μ L application of samples; 10% polyacrylamide gel, electrophoresis 1.5h under 120V, 28mA condition; Pvdf membrane transferring film 3h under 100mA condition; 5% skimmed milk is closed, and drip Nrf2 primary antibodie (1:50), 4 DEG C are spent the night; 5min × 3 time are rinsed with the TBS containing 0.1%Tween 20, add two anti-(1:500) incubated at room temperature 2h of horseradish peroxidase-labeled again, 15min × 3 time are rinsed with 0.1%TBST, ECL luminous detection, X-ray develops, and scan image, records the gray-scale value of each band, with β-actin for after internal reference stdn, compare the change of AFP, Albumin, CK18 expressing quantity.
From table 6, table 7 and Fig. 5, Fig. 6, BMSCs significantly can express liver cell specific AFP, Albumin, CK18 protein expression after HGF and the induction of Rhizome of Bipinnatifid Ginseng saponin(e stimulate differentiation, and be extended for 14 days by 7 days along with the differentiation-inducing time, its expression amount increases further, wherein Rhizome of Bipinnatifid Ginseng saponin(e 200 μ g/mL group action effect and HGF similar.
Table 6 Rhizome of Bipinnatifid Ginseng saponin(e induction BMSCs break up cell AFP, Albumin, CK18 protein expression after 7 days impact (
n=4)
| Group | Dosage | AFP/β-actin | Albumin/β-actin | CK18/β-actin |
| Blank group | ---- | 0.34±0.04 | 0.29±0.01 | 0.26±0.03 |
| HGF group | 20ng/mL | 0.73±0.06** | 0.63±0.03** | 0.44±0.05** |
| Rhizome of Bipinnatifid Ginseng saponin(e | 50μg/mL | 0.45±0.05* | 0.31±0.01 | 0.32±0.03 |
| Rhizome of Bipinnatifid Ginseng saponin(e | 100μg/mL | 0.54±0.07** | 0.38±0.04* | 0.35±0.03* |
| Rhizome of Bipinnatifid Ginseng saponin(e | 200μg/mL | 0.62±0.03** | 0.54±0.06** | 0.43±0.04** |
| Rhizome of Bipinnatifid Ginseng saponin(e+HGF group | 100μg/mL+15ng/mL | 0.85±0.08** | 0.71±0.05** | 0.59±0.07** |
Compare with blank group, * P < 0.05, * * P < 0.01.
Table 7 Rhizome of Bipinnatifid Ginseng saponin(e induction BMSCs break up cell AFP, Albumin, CK18 protein expression after 14 days impact (
n=4)
Compare with blank group, * P < 0.05, * * P < 0.01.
The BMSCs obtained after utilizing separation and purification, peculiar HGF, c-MET, AFP, Albumin, CK18 genetic expression of cell expressing liver cell after can significantly inducing BMSCs to break up after the process of Rhizome of Bipinnatifid Ginseng saponin(e, and present obvious timeliness and dose-effect relationship; Confirm this cell glycogen biosynthesis, picked-up LDL, ureosecretory ability, μ g/mL and positive drug HGF is similar for its neutron ginseng saponin(e 200 simultaneously.Experimental result shows that Rhizome of Bipinnatifid Ginseng saponin(e can obviously induce BMSCs to be divided into liver cell.This research carries out BMSCs transplanting in follow-up body, induces it to be divided into liver cell provide experiment basis with Rhizome of Bipinnatifid Ginseng saponin(e.
Embodiment 3 Rhizome of Bipinnatifid Ginseng saponin(e induction BMSCs goes back to the nest and treats the Effect study of rat liver fibrosis with directed differentiation hepatic lineage
Animal for research: male Sprague-Dawley (SD) rat, quality (200 ± 20) g, is provided by Wuhan University's Experimental Animal Center, credit number: SCXK (Hubei Province) 2008-0004, sub-cage rearing, freely drinks water and ingests.
Test medicine: Rhizome of Bipinnatifid Ginseng medicinal material purchased from shenlongjia, through SanXia University's Natural products research with utilize key lab of Hubei Province Wang Lam to plant the root stock that professor is accredited as araliaceae ginseng plant's Rhizome of Bipinnatifid Ginseng.Rhizome of Bipinnatifid Ginseng saponin(e is by SanXia University's Natural products research and utilize key lab of Hubei Province to provide, and its Rhizome of Bipinnatifid Ginseng saponin content is 92.65%.
SD rat is divided at random normal group, model group, BMSCs transplantation group, Rhizome of Bipinnatifid Ginseng saponin(e group 200mg/kg group, BMSCs transplanting+Rhizome of Bipinnatifid Ginseng saponin(e 200mg/kg group.Except blank group, all the other respectively organize rats by intraperitoneal injection 50%CCl
4(with olive oil) solution, 2 times weekly, volume injected is respectively: 2mL/kg and 1mL/kg, and blank group then injects the sweet oil of respective volume.At injection 50%CCl
4solution, after 2 weeks, respectively injects CCl
4the phenylethyl barbituric acid of 350mg/L is added to increase rat liver to CCl in solution group rat drinking-water
4susceptibility.Giving CCl first
4meanwhile, each group rat oral gavage gives corresponding medicine, and normal group and model group gavage give 0.5% carboxymethylcellulose sodium solution of respective volume, administration every day 1 time, continuous 8 weeks, at the 5th week, and vein transplantation 5 × 10
6the BMSCs of/0.5mLDil mark, the physiological saline of other group injection respective volume, continues administration 4 weeks.In the next day of last administration, after rat uses 20% urethane ester solution (1.2g/kg, i.p) anesthetized rat respectively, open rat abdominal cavity, be separated abdominal cavity aorta, get blood with 10mL disposable syringe, put into the centrifuge tube without antithrombotics, the centrifugal 15min of 3500rpm, separation of serum, gets liver, and weighs its quality, calculate liver index (g/g × 1000), separately get part hepatic tissue Excised Embryos.
1, BMSCs detects at hepatic fibrosis rats distribution in vivo
After BMSCs7 days of vein transplantation Dil mark, random selecting BMSCs transplantation group and BMSCs transplanting+Rhizome of Bipinnatifid Ginseng saponin(e group 200mg/kg group are often organized 2 rats and are carried out BMSCs in the situation analysis of hepatic fibrosis rats distribution in vivo.After putting to death animal, remove liver rapidly, carry out frozen section, then fix with the methyl alcohol of precooling, by the distribution situation of fluorescence microscope BMSCs in Liver fibrosis tissue after buffering glycerine mounting.
2, the Biochemical Indexes of serum
Measure serum alt, AST, ALP, TP, ALB content with automatic clinical chemistry analyzer, and calculate Archon ratio (A/G)=albumins/globulins=albumin/(total protein-albumin).SOD, CAT, GSH-Px, GSH, XOD, T-AOC activity and MDA content in serum is measured by test kit specification sheets marker method.
3, liver histopathology inspection
Hepatic tissue dewaters after 4.0% paraformaldehyde fixes 24 hours, paraffin embedding, makes the section of 4 μm, then carries out HE dyeing, carries out fibrosis classification by improvement Knodell methods of marking.Adopt double-blind method random selecting 5 visuals field to measure collegen filament or reticulin fiber under often opening section 400 times of light microscopics, and calculate its area percentage (reticulin fiber or collegen filament area/hepatic tissue area × 100%).
4, real-time quantitative PCR detects
The strict by specification operation of extraction of hepatic tissue total serum IgE is carried out, and is after cDNA (strictly operating according to Reverse Transcription box) by the RNA reverse transcription of extraction; Add in the reaction tubes through RNase Free process
premix Ex TaqTM II (2X) 12.5 μ L, PCR Forward Primer (10 μm of ol/L) 1 μ L, PCR Reverse Primer (10 μm of ol/L) 1 μ L, DNA profiling 2 μ L, dH
2o (sterile purified water) 8.5 μ L, end reaction system is 25 μ L.Sample is put into real-time quantitative PCR amplification instrument to increase 40 times by 95 DEG C of denaturation 30s, 95 DEG C of sex change 5s, annealing 30s, 72 DEG C of extension 20s conditions.Utilize PCR instrument remittance abroad amplification curve, carry out interpretation of result.
5, Western blot detects
Extract test kit according to tenuigenin-nucleoprotein to illustrate, extract different treatment group myocardial cell's matter albumen and nucleoprotein, after carrying out protein quantification, add sample buffer and boil 5min, respectively get 20 μ L application of samples; 10% polyacrylamide gel, electrophoresis 1.5h under 120V, 28mA condition; Pvdf membrane transferring film 3h under 100mA condition; 5% skimmed milk is closed, and drip Nrf2 primary antibodie (1:50), 4 DEG C are spent the night; 5min × 3 time are rinsed with the TBS containing 0.1%Tween 20, add two anti-(1:500) incubated at room temperature 2h of horseradish peroxidase-labeled again, 10min × 3 time are rinsed with 0.1%TBST, ECL luminous detection, X-ray develops, and scan image, records the gray-scale value of each band, with β-actin for after internal reference stdn, compare the change of its expression amount.
Statistics often organizes data, all with mean ± standard deviation
represent.Data analysis is carried out on SPSS13.0 statistical package, the comparison of many group differences is carried out with one-way analysis of variance (one-way ANOVA), compare the difference of mean between two groups with Dunnett-t inspection, P<0.05 illustrates that difference has statistical significance.
6, Rhizome of Bipinnatifid Ginseng saponin(e impact that BMSCs is raised to Liver Fibrosis Model liver tissues of rats
After Rat Liver Fibrosis Model makes 4 weeks, 3h after modeling, vein transplantation Dil marks BMSCs in heart stalk rat or normal rat body, get liver after 7 days and make frozen section (5 μm), the BMSCs (sending out red fluorescence) that model group only has a small amount of Dil to mark can be observed in hepatic tissue with fluorescence microscope, and with after the treatment of Rhizome of Bipinnatifid Ginseng saponin(e, the BMSCs of a large amount of Dil dye marker.Experimental result shows, Rhizome of Bipinnatifid Ginseng saponin(e significantly can promote the damaged part of BMSCs oriented collection damaged liver as shown in Figure 7, and Fig. 7 is the effect diagram that Rhizome of Bipinnatifid Ginseng saponin(e is raised in Liver Fibrosis Model liver tissues of rats BMSCs.
7, Rhizome of Bipinnatifid Ginseng saponin(e and BMSCs transplant the impact on Liver Fibrosis Model rat body weight and liver index
From table 8, before experiment, before the administration of each group rat, body weight is basically identical, with CCl
4after modeling, its body weight gain speed compared with normal group obviously slows down (P < 0.05), with the improvement had after corresponding pharmacological agent in various degree, but compares with model group and does not have significant difference, and liver is heavy and liver index, occur raising significantly; After alone Rhizome of Bipinnatifid Ginseng saponin(e, BMSCs transplanting and both combined utilization are treated, all improve significantly (P < 0.05 or P < 0.01), wherein obvious with Rhizome of Bipinnatifid Ginseng saponin(e+BMSCs transplantation group action effect.
Table 8 Rhizome of Bipinnatifid Ginseng saponin(e and BMSCs transplant impact on Liver Fibrosis Model rat body weight and liver index (
n=8)
Compare with normal group,
#p < 0.05,
##p < 0.01 compares with model group, * P < 0.05, * * P < 0.01
8, Rhizome of Bipinnatifid Ginseng saponin(e and BMSCs transplant the impact on Liver Fibrosis Model rat liver fibrosis
From table 9 and Fig. 8, normal group liver lobule structure is normal, and liver rope queueing discipline is orderly, sinus hepaticus and portal area inoblast few; Model group liver rope arrangement disorder, liver cell diffusivity steatosis, portal area and the interior visible significantly inflammatory cell infiltration of liver lobule, proliferation of fibrous tissue is obvious; Alone Rhizome of Bipinnatifid Ginseng saponin(e, BMSCs transplant and after both combined utilization treatments, hepatocellular degeneration alleviates, and inflammatory cell infiltration is less, and collegen filament, reticulin fiber deposition all obviously reduces.Statistical study finds, alone Rhizome of Bipinnatifid Ginseng saponin(e, BMSCs transplant and the liver fibrosis classification of both combined utilization groups, collegen filament area ratio, reticulin fiber area ratio and Hyp content more all have significant difference (P < 0.05 or P < 0.01) with model group, wherein obvious with Rhizome of Bipinnatifid Ginseng saponin(e+BMSCs transplantation group action effect.
Table 9 Rhizome of Bipinnatifid Ginseng saponin(e and BMSCs transplant impact on Liver Fibrosis Model rat liver fibrosis (
n=8)
Compare with normal group,
#p < 0.05,
##p < 0.01 compares with model group, * P < 0.05, * * P < 0.01
9, Rhizome of Bipinnatifid Ginseng saponin(e and BMSCs transplant the impact on Liver Fibrosis Model Liver Function
From table 10, model group rats serum AST, ALT and ALP content obviously raise, and TP, ALB level and A/G ratio significantly reduce, and compare have significant difference (P<0.01) with normal group; After alone Rhizome of Bipinnatifid Ginseng saponin(e, BMSCs transplanting and both combined utilization are treated, the liver function index of above-mentioned abnormal change is effectively reversed, and present good dose-effect relationship, compare with model group and there is significant difference (P<0.05 or P<0.01), wherein obvious with Rhizome of Bipinnatifid Ginseng saponin(e+BMSCs transplantation group action effect.
Table 10 Rhizome of Bipinnatifid Ginseng saponin(e and BMSCs transplant impact on Liver Fibrosis Model Liver Function (
n=8)
Compare with normal group,
#p < 0.05,
##p < 0.01 compares with model group, * P < 0.05, * * P < 0.01
10, Rhizome of Bipinnatifid Ginseng saponin(e and BMSCs transplant the impact on Liver Fibrosis Model rat blood serum oxidation index
From table 11: model group rats SOD in serum, CAT and GSH-Px content obviously reduce, and MDA level significantly raises, compare with normal group and there is significant difference (P<0.01); After alone Rhizome of Bipinnatifid Ginseng saponin(e, BMSCs transplanting and both combined utilization are treated, the oxidation index of above-mentioned abnormal change is effectively reversed, and present good dose-effect relationship, compare with model group and there is significant difference (P<0.05 or P<0.01), wherein obvious with Rhizome of Bipinnatifid Ginseng saponin(e+BMSCs transplantation group action effect.
Table 11 Rhizome of Bipinnatifid Ginseng saponin(e and BMSCs transplant impact on Liver Fibrosis Model rat blood serum oxidation index (
n=8)
Compare with normal group,
#p < 0.05,
##p < 0.01 compares with model group, * P < 0.05, * * P < 0.01
11, Rhizome of Bipinnatifid Ginseng saponin(e and BMSCs transplant the impact on Liver Fibrosis Model liver tissues of rats mesostroma cell derived factor-1 and Receptor Gene Expression thereof
As shown in Table 12, model group hepatic tissue mesostroma cell derived factor-1 and the horizontal compared with normal group of acceptor (SDF-1 and CXCR4) mrna expression thereof increase to some extent, but compare with normal group and do not have significant difference (P>0.05); After alone Rhizome of Bipinnatifid Ginseng saponin(e, BMSCs transplanting and both combined utilization are treated, its expression level obviously increases, and present good dose-effect relationship, compare with model group, there is significant difference (P<0.05 or P<0.01), wherein obvious with Rhizome of Bipinnatifid Ginseng saponin(e+BMSCs transplantation group action effect.
Table 12 Rhizome of Bipinnatifid Ginseng saponin(e and BMSCs transplant impact on Liver Fibrosis Model liver tissues of rats mesostroma cell derived factor-1 and Receptor Gene Expression thereof (
n=4)
| Group | Dosage (mg/kg) | SDF-1/GAPDH | CXCR4/GAPDH |
| Normal group | --- | 0.29±0.04 | 0.48±0.03 |
| Model group | --- | 0.33±0.02 # | 0.59±0.04 |
| BMSCs transplantation group | --- | 0.59±0.02* | 0.78±0.03* |
| Rhizome of Bipinnatifid Ginseng saponin(e group | 200 | 0.71±0.04** | 0.84±0.02** |
| BMSCs transplanting+Rhizome of Bipinnatifid Ginseng saponin(e group | 200 | 0.76±0.03** | 0.90±0.03** |
Compare with normal group,
#p < 0.05,
##p < 0.01 compares with model group, * P < 0.05, * * P < 0.01
12, Rhizome of Bipinnatifid Ginseng saponin(e and BMSCs transplant the impact on HGF, c-MET mrna expression in Liver Fibrosis Model liver tissues of rats
As shown in Table 13, in model group hepatic tissue, the horizontal compared with normal group of HGF, c-MET mrna expression increases to some extent, but compares with normal group and do not have significant difference (P>0.05); After alone Rhizome of Bipinnatifid Ginseng saponin(e, BMSCs transplanting and both combined utilization are treated, its expression level obviously increases, and present good dose-effect relationship, compare with model group, there is significant difference (P<0.05 or P<0.01), wherein obvious with Rhizome of Bipinnatifid Ginseng saponin(e+BMSCs transplantation group action effect.
Table 13 Rhizome of Bipinnatifid Ginseng saponin(e and BMSCs transplant impact on HGF, c-MET mrna expression in Liver Fibrosis Model liver tissues of rats (
n=4)
| Group | Dosage (mg/kg) | HGF/GAPDH | c-MET/GAPDH |
| Normal group | --- | 0.31±0.04 | 0.29±0.03 |
| Model group | --- | 0.35±0.03 | 0.32±0.02 |
| BMSCs transplantation group | --- | 0.43±0.01* | 0.39±0.01* |
| Rhizome of Bipinnatifid Ginseng saponin(e group | 200 | 0.47±0.04** | 0.46±0.03** |
| BMSCs transplanting+Rhizome of Bipinnatifid Ginseng saponin(e group | 200 | 0.69±0.02** | 0.67±0.04** |
Compare with normal group,
#p < 0.05,
##p < 0.01 compares with model group, * P < 0.05, * * P < 0.01
13, Rhizome of Bipinnatifid Ginseng saponin(e and BMSCs transplant the impact on Liver Fibrosis Model liver tissues of rats SOD, GPX1 and CAT mrna expression
From table 14: in model group hepatic tissue, SOD1, SOD2, SOD3, GPX1 and CAT mrna expression obviously declines, compare with normal group and there is significant difference (P<0.01); After alone Rhizome of Bipinnatifid Ginseng saponin(e, BMSCs transplanting and both combined utilization are treated, its mrna expression level be improved significantly, compare with model group and there is significant difference (P<0.05 or P<0.01), wherein obvious with Rhizome of Bipinnatifid Ginseng saponin(e+BMSCs transplantation group action effect.
Table 14 Rhizome of Bipinnatifid Ginseng saponin(e and BMSCs transplant impact on Liver Fibrosis Model liver tissues of rats SOD, GPX1 and CAT mrna expression (
n=4)
Compare with normal group,
#p < 0.05,
##p < 0.01 compares with model group, * P < 0.05, * * P < 0.01
14, Rhizome of Bipinnatifid Ginseng saponin(e and BMSCs transplant the impact expressed Liver Fibrosis Model liver tissues of rats α-SMA, MMP-1 and TIMP-1mRNA
In order to confirm that CCl4 causes the relation of liver injury and hepatic fibrosis further, we use the Real-time PCR Analysis mrna expression of α-SMA, MMP-1 and TIMP-1.From table 15, in model group hepatic tissue, the mrna expression of α-SMA, MMP-1 and TIMP-1 obviously declines, and compares have significant difference (P<0.01) with normal group; After alone Rhizome of Bipinnatifid Ginseng saponin(e, BMSCs transplanting and both combined utilization are treated, α-SMA and TIMP-1mRNA expression level are obviously reduced, MMP1 obviously raises, compare with model group and there is significant difference (P<0.05 or P<0.01), wherein obvious with Rhizome of Bipinnatifid Ginseng saponin(e+BMSCs transplantation group action effect.
Table 15 Rhizome of Bipinnatifid Ginseng saponin(e and BMSCs transplant Liver Fibrosis Model liver tissues of rats α-SMA, MMP-1 and TIMP-1mRNA are expressed impact (
n=4)
Compare with normal group,
#p < 0.05,
##p < 0.01 compares with model group, * P < 0.05, * * P < 0.01
15, Rhizome of Bipinnatifid Ginseng saponin(e and BMSCs transplant the impact on Liver Fibrosis Model liver tissues of rats Nrf2 protein expression
From table 16 and Fig. 9, in each experimental rat hepatic tissue cell matter, Nrf2 protein expression level is without significant difference.In hepatic tissue cell core, in visible normal group nucleus, Nrf2 expresses less, and in model group karyon, Nrf2 expresses compared with normal group increases to some extent, but does not have significant difference (P > 0.05); After alone Rhizome of Bipinnatifid Ginseng saponin(e, BMSCs transplanting and both combined utilization are treated, Nrf2 protein expression level in nucleus can be made obviously to raise, compare with model group and there is significant difference (P<0.05 or P<0.01), wherein obvious with Rhizome of Bipinnatifid Ginseng saponin(e+BMSCs transplantation group action effect.
Table 16 Rhizome of Bipinnatifid Ginseng saponin(e and BMSCs transplant impact on Liver Fibrosis Model liver tissues of rats Nrf2 protein expression (
n=4)
| Group | Dosage (mg/kg) | Tenuigenin Nrf2/ β-actin | Nucleus Nrf2/ β-actin | |
| Normal group | Normal group | 0.38±0.04 | 0.13±0.02 | |
| Model group | Model group | 0.41±0.05 | 0.15±0.03 | |
| BMSCs transplantation group | Rhizome of Bipinnatifid Ginseng saponin(e group | 100 | 0.39±0.04 | 0.19±0.02* |
| Rhizome of Bipinnatifid Ginseng saponin(e group | Rhizome of Bipinnatifid Ginseng saponin(e group | 200 | 0.43±0.03 | 0.21±0.03** |
| BMSCs transplanting+Rhizome of Bipinnatifid Ginseng saponin(e group | Colchicine group | 100 | 0.46±0.02 | 0.34±0.04** |
Compare with normal group,
#p < 0.05,
##p < 0.01 compares with model group, * P < 0.05, * * P < 0.01
Found through experiments, Rhizome of Bipinnatifid Ginseng saponin(e obviously can improve liver function, reduce ALT, AST, ALP, XOD and MDA level in blood, increase active and TP, ALB level of SOD, GSH-Px, CAT, T-AOC and A/G ratio, reduce liver fibrosis classification, collegen filament area ratio, reticulin fiber area ratio and Hyp content, improve liver organization morphology; Raise SOD1, SOD2, SOD3, CAT, GPX1 and MMP1 genetic expression and lower α-SMA and TIMP-1 genetic expression, promoting the displacement of Nrf2 core, increase the protein expression level of Nrf2 in liver cell nuclear.Experimental result shows, Rhizome of Bipinnatifid Ginseng saponin(e is to CCl
4cause rat liver fibrosis and have provide protection, promote the displacement of Nrf2 core, strengthen endogenous anti-oxidative system function, and then suppress hepatic fibrosis, recovering the normal physiological function of liver may be one of its mechanism of action.This research is provided fundamental basis and experimental basis for the clinical control for hepatic fibrosis of Rhizome of Bipinnatifid Ginseng saponin(e, simultaneously also for provide possibility with BMSCs combined utilization.
Although give some embodiments of the present invention, it will be understood by those of skill in the art that without departing from the spirit of the invention herein, can change embodiment herein.Above-described embodiment is exemplary, should using embodiment herein as the restriction of interest field of the present invention.
Claims (10)
1. the hepatocellular application of Rhizome of Bipinnatifid Ginseng saponin(e differentiation of stem cells, is characterized in that, the Rhizome of Bipinnatifid Ginseng saponin(e extracted from Rhizome of Bipinnatifid Ginseng is application in liver cell inductor preparing differentiation of stem cells.
2. the hepatocellular application of Rhizome of Bipinnatifid Ginseng saponin(e differentiation of stem cells as claimed in claim 1, it is characterized in that, described stem cell is adult stem cell.
3. the hepatocellular application of Rhizome of Bipinnatifid Ginseng saponin(e differentiation of stem cells as claimed in claim 2, it is characterized in that, described adult stem cell is mesenchymal stem cells MSCs.
4. the hepatocellular application of Rhizome of Bipinnatifid Ginseng saponin(e differentiation of stem cells as claimed in claim 3, it is characterized in that, described differentiation of stem cells is that the composition of liver cell inductor comprises: HG-DMEM, penicillin, Streptomycin sulphate, glutamine, foetal calf serum, Rhizome of Bipinnatifid Ginseng saponin(e alone or with the coupling of Rhizome of Bipinnatifid Ginseng saponin(e pHGF.
5. the method for the hepatocellular application of a Rhizome of Bipinnatifid Ginseng saponin(e differentiation of stem cells as claimed in claim 1, it is characterized in that, get rat bone marrow mesenchymal stem cells and carry out Secondary Culture, get the stable mesenchymal stem cells MSCs gone down to posterity and carry out inducing culture in inducing culture, obtain ripe hepatocyte-like cells; Described inducing culture is the basic medium being added with Rhizome of Bipinnatifid Ginseng saponin(e and cell growth factor, in described basic medium, the addition of Rhizome of Bipinnatifid Ginseng saponin(e and cell growth factor is: the concentration of Rhizome of Bipinnatifid Ginseng saponin(e is: 50-200 μ g/mL, cell growth factor concentration is: 10-30ng/mL.
6. the hepatocellular application method of Rhizome of Bipinnatifid Ginseng saponin(e differentiation of stem cells as claimed in claim 5, it is characterized in that, the concentration of the cell growth factor added in described basic medium is: 20ng/mL.
7. the hepatocellular application method of Rhizome of Bipinnatifid Ginseng saponin(e differentiation of stem cells as claimed in claim 5, it is characterized in that, get rat bone marrow mesenchymal stem cells and carry out Secondary Culture, get the mesenchymal stem cells MSCs of the third generation, be inoculated in low sugar DMEM substratum, after described mesenchymal stem cells MSCs individual layer grows to 80%, the substratum of described mesenchymal stem cells MSCs is replaced by the inducing culture containing Rhizome of Bipinnatifid Ginseng saponin(e and cell growth factor and carries out inducing culture, within every 3 days, change liquid 1 time, after cultivating 2 weeks, obtain ripe hepatocyte-like cells.
8. the hepatocellular inductor of differentiation of stem cells, is characterized in that, described inductor contains Rhizome of Bipinnatifid Ginseng saponin(e.
9. Rhizome of Bipinnatifid Ginseng saponin(e treatment hepatopathy in an application, it is characterized in that, described in be applied as Rhizome of Bipinnatifid Ginseng saponin(e treatment hepatopathy in pharmaceutical composition in application.
10. treat the pharmaceutical composition in hepatopathy, it is characterized in that, containing Rhizome of Bipinnatifid Ginseng saponin(e in the pharmaceutical composition in described treatment hepatopathy; The dosage form of described pharmaceutical composition comprises: injection, tablet, capsule, aerosol, suppository, film, pill, ointment, control-released agent, sustained release dosage or nanometer formulation.
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| CN109295080A (en) * | 2018-09-19 | 2019-02-01 | 昆明理工大学 | Panax japonicus majoris β-amyrin synthase gene Pj β-AS purposes |
| CN111235100A (en) * | 2020-02-21 | 2020-06-05 | 新乡医学院 | Culture method of human umbilical cord blood mesenchymal stem cells |
| CN118389414A (en) * | 2024-04-24 | 2024-07-26 | 广州卓悦生物科技有限公司 | Method for promoting osteoblast differentiation of mesenchymal stem cells |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102134268A (en) * | 2011-01-14 | 2011-07-27 | 陕西中医学院 | Method for preparing panax japonicus saponin IVa and application of panax japonicus saponin IVa in preparing a medicament for protecting liver and lowering transaminase |
| CN103045531A (en) * | 2011-10-14 | 2013-04-17 | 北京清美联创干细胞科技有限公司 | Application of ginsenoside Rd for inducing mesenchymal stem cell to be directionally differentiated in to hepatic cell in vitro |
-
2015
- 2015-06-29 CN CN201510366605.7A patent/CN104962515A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102134268A (en) * | 2011-01-14 | 2011-07-27 | 陕西中医学院 | Method for preparing panax japonicus saponin IVa and application of panax japonicus saponin IVa in preparing a medicament for protecting liver and lowering transaminase |
| CN103045531A (en) * | 2011-10-14 | 2013-04-17 | 北京清美联创干细胞科技有限公司 | Application of ginsenoside Rd for inducing mesenchymal stem cell to be directionally differentiated in to hepatic cell in vitro |
Non-Patent Citations (7)
| Title |
|---|
| 刘志锋等: "骨髓干细胞定向分化为肝样细胞的研究进展", 《世界华人消化杂志》 * |
| 姜涛: "骨髓间充质干细胞分化为肝样细胞治疗HBV小鼠肝损伤的实验研究", 《中国优秀硕士学位论文全文数据库医药卫生科技辑》 * |
| 张继红等: "珠子参皂苷与ＢＭＳＣｓ联用治疗大鼠肝纤维化作用研究", 《三峡大学学报（自然科学版）》 * |
| 张继红等: "珠子参皂苷对四氯化碳致大鼠肝纤维化的保护作用", 《中药药理与临床》 * |
| 张继红等: "珠子参皂苷诱导骨髓干细胞分化为肝样细胞的体外实验研究", 《中药药理与临床》 * |
| 王薇等: "珠子参总皂苷对四氯化碳致大鼠慢性肝损伤的保护作用", 《中药药理与临床》 * |
| 赵东东等: "珠子参叶的皂苷成分研究", 《中南药学》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106237313A (en) * | 2016-09-30 | 2016-12-21 | 广州赛莱拉干细胞科技股份有限公司 | A kind of umbilical cord mesenchymal stem cells injection and its preparation method and application |
| CN109295080A (en) * | 2018-09-19 | 2019-02-01 | 昆明理工大学 | Panax japonicus majoris β-amyrin synthase gene Pj β-AS purposes |
| CN111235100A (en) * | 2020-02-21 | 2020-06-05 | 新乡医学院 | Culture method of human umbilical cord blood mesenchymal stem cells |
| CN111235100B (en) * | 2020-02-21 | 2022-04-29 | 新乡医学院 | Culture method of human umbilical cord blood mesenchymal stem cells |
| CN118389414A (en) * | 2024-04-24 | 2024-07-26 | 广州卓悦生物科技有限公司 | Method for promoting osteoblast differentiation of mesenchymal stem cells |
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