CN108210518B - Combination of umbilical cord mesenchymal stem cells for treating hyperglycemia and diabetic nephropathy - Google Patents
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Abstract
The invention provides an application of umbilical cord mesenchymal stem cells combined with a traditional Chinese medicine extract in preparing a medicine for treating hyperglycemia and diabetic nephropathy. In the invention, the umbilical cord mesenchymal stem cells with differentiation capacity and potential for treating diabetes and the traditional Chinese medicine extract with the effects of reducing blood sugar, improving the state of kidney and inhibiting the kidney disease caused by diabetes are combined to prepare the medicine for treating hyperglycemia and diabetic nephropathy, so that the blood sugar can be effectively controlled, and the function of the kidney can be improved.
Description
Technical Field
The invention relates to the field of medicines for treating hyperglycemia and diabetic nephropathy, in particular to application of umbilical cord mesenchymal stem cells in combination with various traditional Chinese medicinal materials related to diabetes treatment in preparation of medicines for treating hyperglycemia and diabetic nephropathy.
Background
According to the World Health Organization (WHO) projection, by 2030, diabetic patients will increase from two hundred million to five hundred million to four hundred million and nine million in 2010. Diabetes is also one of the most serious diseases which endanger the health of people in China, and various acute and chronic complications of diabetes are the main reasons for death of diabetics. Diabetic nephropathy is one of the major microvascular pathological complications of diabetic patients, and is also the main cause of end-stage renal failure, and how to effectively prevent and treat diabetic nephropathy is still one of the important subjects facing diabetes research.
Pathophysiological characteristics of diabetic nephropathy: microalbuminuria is the earliest clinical manifestation of diabetic nephropathy kidney injury, the formation of which is primarily associated with histological changes in the kidney, such as extracellular matrix deposition, thickening of the glomerular basement membrane, and expansion of the glomerular mesangium. Massive proteinuria will occur later in the course of diabetic nephropathy, glomerular filtration rate will decrease progressively, and renal histological changes include glomerular sclerosis, tubular interstitial fibrosis, and arterial transparence.
There are many current treatments for diabetic nephropathy, including blood glucose control, blood pressure control, and proteinuria reduction. End-stage patients may have renal replacement therapy available, including hemodialysis and kidney transplantation, which is only effective if islet B-cell transplantation is performed simultaneously, otherwise renal failure will reoccur. Hemodialysis has a major impact on the quality of life of patients, and kidney transplantation is the most effective means for treating end-stage renal disease today. However, the shortage of kidney sources greatly limits the spread of kidney transplantation. The kidney is an terminally differentiated organ with far less regenerative potential than other organs of the human body. Kidney function is not performed by a single cell, but is performed by constituting a unit with cells having different functions, which also makes the regeneration of the kidney difficult.
Mesenchymal Stem Cells (MSCs), also known as multipotent Mesenchymal stromal cells, are non-hematopoietic adult stem cells with a wide tissue distribution and self-renewal and differentiation capacity. MSCs were first isolated from bone marrow and subsequently obtained from other tissues and organs including hair follicles, dental roots, brain periosteum, perichondrium, dermis, cord blood, umbilical cord, placenta, adipose muscle, lung, liver and spleen. Several studies have shown that MSCs cultured in vitro specifically express 113 transcripts and 17 proteins that hematopoietic cells do not express. The minimum criteria for human MSCs were defined according to the international commission on cell therapy (ISCT) by the subordinate committee on mesenchymal and tissue stem cells: a) under standard culture conditions, MSCs must have adhesion to plastic substrates; b) positive for CD105, CD73, CD90, negative for CD45, CD34, CD14 or CD11b, CD79a or CD19 and HLA-DR; c) MSCs can differentiate into osteoblasts, adipocytes and chondrocytes under standard differentiation conditions in vitro.
Mesenchymal stem cells have a plurality of exploratory researches in various disciplines, and basic researches prove that kidney cells have regeneration potential recently, and the stem cells have a plurality of exploratory researches on treating kidney injury including diabetic nephropathy. Recently, Umbilical Cord Mesenchymal Stem Cells (UCMSCs) have been increasingly highlighted by researchers. Umbilical cord mesenchymal stem cells have the ability to self-renew and differentiate into different cells. The umbilical cord mesenchymal stem cells are derived from umbilical cord. The umbilical cord is the waste after the delivery of the fetus, has wide sources and does not have ethical problems. In addition, due to the existence of the fetal blood barrier, the probability of virus pollution to the umbilical cord is obviously reduced, and the material taking process is painless. More importantly, the umbilical cord mesenchymal stem cells can still maintain the characteristics of the stem cells after continuous and repeated passages, are easy to culture for a long time and can be frozen, and have high proliferation capacity, safety and effectiveness. Human umbilical cord mesenchymal stem cells may be present in mice for a considerable period of time without eliciting an immune response in the host with a single injection. The characteristic that the umbilical cord mesenchymal stem cells are differentiated into specific functional cells is utilized for transplantation, and the cells with irreversible loss of functions in vivo can be replaced, so that the umbilical cord mesenchymal stem cells have wide clinical application prospects in the aspect of treatment of diseases such as tumors, diabetes, blood diseases, myocardial necrosis, Parkinson's disease, spinal cord injury and the like, which do not have effective treatment means at present. The application of stem cell technology to the treatment of experimental diabetic nephropathy has made great progress in experimental animals and is likely to become one of the effective methods for treating diabetes in the future.
The traditional Chinese medicinal materials have the effects of strengthening body resistance and tonifying qi, and modern pharmaceutical research shows that various traditional Chinese medicinal materials such as astragalus, medlar, kudzu root, schisandra chinensis, salvia miltiorrhiza and the like have various active ingredients, have long medicinal history and are common traditional Chinese medicine types for treating diabetes in traditional Chinese medicine of past generations.
The research concept of Chinese medicine for preventing and treating diabetes mellitus develops from the traditional empirical medicine with clinical experience to the medicine with more scientific evidence. At present, a great amount of blood sugar reducing substances are found from natural traditional Chinese medicine plants, and mainly comprise polysaccharide, flavone, alkaloid and the like. Alkaloid compounds are of great interest due to their good hypoglycemic effects, and in the last two decades, a large number of plant alkaloids have been extracted and separated, and thousands of alkaloids separated at present have pharmacological activity for reducing blood sugar, and have been widely used in the medical field. However, there is still a lack of thorough understanding of the mechanism of absorption and metabolism of physiological functions, the mechanism of active group inhibition, and the mechanism of stabilization.
The present western medicine for treating diabetes, aiming at the etiology, improves insulin resistance and protects the functions of pancreas β cells, selects the medicines capable of improving insulin resistance, mainly comprises insulin sensitizers, can cause edema and weight gain of patients after long-term administration of the sensitizers, and serious liver damage of few patients.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention provides application of umbilical cord mesenchymal stem cells combined with various traditional Chinese medicine extracts in preparation of a medicine for treating hyperglycemia and diabetic nephropathy. In the invention, the umbilical cord mesenchymal stem cells with differentiation capacity and potential for treating diabetes and a plurality of traditional Chinese medicine extracts with the effects of reducing blood sugar, improving the state of kidney and inhibiting the kidney disease caused by diabetes are combined to prepare the medicine for treating hyperglycemia and diabetic nephropathy, so that the blood sugar level can be effectively controlled, and the function of the kidney can be improved.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
the umbilical cord mesenchymal stem cells are combined with various traditional Chinese medicine extracts to prepare the medicine for treating hyperglycemia and diabetic nephropathy.
In the invention, the mesenchymal stem cells are combined with various traditional Chinese medicine extracts, so that the differentiation capacity of the mesenchymal stem cells can be utilized to supplement damaged cells; meanwhile, the blood sugar level can be further effectively controlled by using various traditional Chinese medicine extracts, and meanwhile, the kidney can be protected to a certain extent, so that the kidney function is effectively improved.
In the present invention, the umbilical cord mesenchymal stem cells are human umbilical cord mesenchymal stem cells.
Optionally, in the present invention, the application further includes a step of culturing umbilical cord mesenchymal stem cells, and then applying the umbilical cord mesenchymal stem cells obtained by the culture in combination with a plurality of traditional Chinese medicine extracts.
Optionally, in the invention, the culturing is performed by primary umbilical cord mesenchymal stem cells, or after the umbilical cord mesenchymal stem cells are recovered from the frozen umbilical cord mesenchymal stem cells, culturing in a culture medium, and then performing subculture.
Optionally, in the invention, the culture medium is DMEM/F12 culture medium containing FBS, penicillin and streptomycin.
Optionally, in the invention, the content of the FBS is 10%, and the concentration of the penicillin and the streptomycin is 100U/ml.
Alternatively, in the present invention, the subculture is performed in an incubator containing 5% CO 2.
Optionally, in the invention, the temperature of the subculture is 35-38 ℃; preferably, in the invention, the temperature of the subculture is 36-37 ℃.
Optionally, in the present invention, the application further includes a step of digesting the umbilical cord mesenchymal stem cells obtained by subculture to obtain stably proliferating cells, detecting, and then applying in combination with a plurality of traditional Chinese medicine extracts.
Alternatively, in the present invention, the digestion is with trypsin.
Optionally, in the invention, the concentration of the trypsin is 0.20-0.25%; preferably, in the present invention, the concentration of trypsin is 0.24% to 0.25%.
Optionally, in the present invention, the multiple traditional Chinese medicine extracts are extracts of one or more of astragalus, ginseng, codonopsis pilosula, salvia miltiorrhiza, panax notoginseng, lycium barbarum, pueraria lobata, schisandra chinensis, atractylodes macrocephala, semen cassiae, radix rehmanniae recen and polygonatum sibiricum.
Optionally, in the present invention, the plurality of traditional Chinese medicine extracts are extracts of one or more of astragalus, ginseng, codonopsis pilosula, salvia miltiorrhiza, panax notoginseng, lycium barbarum, pueraria lobata, schisandra chinensis, atractylodes macrocephala and cassia seed.
Optionally, the present invention is characterized in that the plurality of traditional Chinese medicine extracts are extracts of one or more of astragalus, ginseng, salvia miltiorrhiza, panax notoginseng, atractylodes macrocephala koidz and schisandra chinensis.
Optionally, in the invention, the multiple traditional Chinese medicine extracts are extracts of any 6, 5, 4, 3 and 2 of astragalus, ginseng, salvia miltiorrhiza, pseudo-ginseng, bighead atractylodes rhizome and schisandra chinensis.
Optionally, in the invention, the multiple traditional Chinese medicine extracts are one or more of astragalus, ginseng and salvia miltiorrhiza, and extracts of pseudo-ginseng and bighead atractylodes rhizome are added.
Optionally, in the invention, the multiple traditional Chinese medicine extracts are one or more of astragalus, ginseng and salvia miltiorrhiza, and extracts of pseudo-ginseng, bighead atractylodes rhizome and schisandra chinensis are added.
Optionally, the present invention is characterized in that the plurality of traditional Chinese medicine extracts are extracts of salvia miltiorrhiza, panax notoginseng and atractylodes macrocephala.
The concentration of the traditional Chinese medicine extract suitable for the proliferation of the huMSCs and HMC is as follows: 0.1-10mg/ml of astragalus root, 0.1-10mg/ml of salvia miltiorrhiza and 0.1-10mg/ml of ginseng; preferably, the astragalus, the salvia miltiorrhiza and the ginseng can also be respectively selected from 0.2, 0.5, 0.8, 1, 1.5, 2, 3, 4, 5, 7 and 9mg/ml for culturing the humSCs or/and HMC to proliferate. The above Chinese medicinal extract culture reagents can be used for culturing cells respectively or in combination. The above Chinese medicinal extract culture reagent can be mixed with the following Chinese medicinal extracts for cell culture: adding fructus Schisandrae 20-150ul/ml and/or Atractylodis rhizoma 10-50ul/ml for culturing humSCs to proliferate; the preferred schisandra can also select 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140ul/ml and other similar concentrations as the traditional Chinese medicine extract additive, and the atractylodes can select 20, 30, 40ul/ml and other similar concentrations as the traditional Chinese medicine extract additive, and is co-cultured with the huMSCs or/and the HMC.
The added amount and concentration of the traditional Chinese medicine extract are obtained by adopting the following extraction method:
slicing Ginseng radix, Saviae Miltiorrhizae radix, and radix astragali, filtering, oven drying, grinding, and sieving with 80-120 mesh sieve to obtain fine powder;
pulverizing fructus Schisandrae chinensis and Notoginseng radix, placing into a container, soaking in 6-16 times of 50-80% ethanol solution, heating and reflux extracting for 3 times, each for 1-6 hr, filtering, mixing filtrates, distilling under reduced pressure to remove ethanol, and concentrating to obtain concentrated solution with relative density of 1.10-1.13 at 60 deg.C;
decocting Atractylodis rhizoma with 12 times, 10 times and 8 times of water for three times, each for 2 hr, mixing the three water extractive solutions, and vacuum concentrating under reduced pressure to 1g medicinal material/ml extractive solution for use.
Compared with the prior art, the invention has the beneficial effects that:
in the invention, the umbilical cord mesenchymal stem cells with differentiation capacity and potential for treating diabetes and a plurality of traditional Chinese medicine extracts with the effects of reducing blood sugar, improving the state of kidney and inhibiting the kidney disease caused by diabetes are combined to prepare the medicine for treating hyperglycemia and diabetic nephropathy, so that the blood sugar level can be effectively controlled, and the function of the kidney can be improved.
The method comprises the steps of selecting main medicines with more than ten flavors from traditional Chinese medicine formulas related to diabetes treatment, combining extracts of the main medicines, and screening out a traditional Chinese medicine extract composition which can be used in cooperation with umbilical cord mesenchymal stem cells and has the effects of reducing blood sugar and improving kidney state.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
Fig. 1 shows the cell morphology and staining of human mesenchymal stem cells at different stages under a microscope, wherein a is the cell morphology observed under a 25 × microscope for human mesenchymal stem cells without induced differentiation treatment; b is the cell staining condition of the human mesenchymal stem cells observed under a 10X microscope after induced differentiation treatment by the adipogenic induction reagent; and C is the cell staining condition of the human mesenchymal stem cells under a microscope of 10 multiplied by the weight after the differentiation induction treatment by the osteogenesis inducing reagent.
FIG. 2 is a proliferation chart of human umbilical cord mesenchymal stem cells (humSCs) under different concentrations of herbal extracts. a is astragalus; b is ginseng; c is salvia miltiorrhiza; d is pseudo-ginseng; e is largehead atractylodes rhizome; f is fructus Schisandrae.
FIG. 3 is a graph showing the proliferation of glomerular cells (HMC) under different conditions with different concentrations of herbal extracts. a is astragalus; b is ginseng; c is salvia miltiorrhiza; d is pseudo-ginseng; e is largehead atractylodes rhizome; f is fructus Schisandrae.
Fig. 4 is a proliferation chart of co-culture of human umbilical cord mesenchymal stem cells and HMC under the condition of different combined traditional Chinese medicine extracts.
FIG. 5 is a graph showing the content of collagen in supernatants FN and IV detected by ELISA when human umbilical cord mesenchymal stem cells and various herbal extracts were mixed and cultured in humSCs.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Example 1 human umbilical cord mesenchymal stem cell culture and passage method
The humSCs (namely human umbilical cord mesenchymal stem cells) are placed in DMEM/F12 medium containing 10% FBS, 100U/ml penicillin and streptomycin after primary culture; then, carrying out conventional culture and passage in an incubator at 37 ℃ and 5% CO 2; the cells obtained by subculture were digested with 0.25% trypsin, and stably proliferating cells were collected.
Will 106The collected human umbilical cord mesenchymal stem cells were suspended in 100ml of PBS buffer solution containing 1% bovine serum albumin, and the expression of positive antibodies (CD90, CD105, CD73) and negative antibodies (CD45, CD34, CD11b, CD19 and HLA-DR) was examined by flow cytometry. The results are shown in FIG. 1, which shows that the cultured human umbilical cord mesenchymal stem cells meet the stem cell standard.
Example 2 preparation of extracts of Chinese herbs
The Chinese medicinal extracts are prepared according to the following method:
slicing Ginseng radix, Saviae Miltiorrhizae radix, and radix astragali, filtering, oven drying, grinding, and sieving with 80-120 mesh sieve to obtain fine powder.
Pulverizing fructus Schisandrae chinensis and Notoginseng radix, placing into a container, soaking in 6-16 times volume of 50-80% ethanol solution for 1-10 hr, heating and reflux extracting for 3 times, each for 1-6 hr, filtering, mixing filtrates, distilling under reduced pressure to remove ethanol, and concentrating to obtain concentrated solution with relative density of 1.10-1.13 at 60 deg.C.
Decocting Atractylodis rhizoma with 12 times, 10 times and 8 times of water for three times, each for 2 hr, mixing the three water extractive solutions, and vacuum concentrating under reduced pressure to 1g medicinal material/ml extractive solution for use.
Experimental example 3 Effect of Chinese medicinal extracts on growth of human umbilical cord mesenchymal stem cells and glomerular cells
Selecting humSCs (human umbilical cord mesenchymal stem cells) and HMC (glomerular cells) with good growth state at a ratio of 3 × 103Inoculating each well in a 96-well plate, adding traditional Chinese medicine extracts with different concentrations for co-culture after cells adhere to the wall, adding no traditional Chinese medicine extract in a control group, adding no cells in a zero-setting hole, only adding 100 mu L of culture solution, making 5 multiple wells in parallel in each group, adding 10 mu LCCK8 solution in each group after culturing for 4h,24h and 48h, and selecting 490nm wavelength to measure the absorbance (OD) value of each well after incubating for 4h at 37 ℃. Root of herbaceous plantEffective concentration measured according to cck-8 results. The test results are shown in fig. 2 and 3.
According to the detection result, the astragalus, the salvia miltiorrhiza and the ginseng traditional Chinese medicine extract can effectively promote the proliferation of the huMSCs and the HMC, and cannot generate the inhibiting or toxic effect on the huMSCs and the HMC; while the schisandra chinensis and the bighead atractylodes rhizome hardly have influence on the proliferation of the huMSCs and the HMC, the pseudo-ginseng has an obvious inhibiting effect on the proliferation of the huMSCs and the HMC compared with a control group.
Example 4 Effect of the Mixed combination of different herbal extracts on the growth of human umbilical cord mesenchymal Stem cells
The culture and detection method is the same as example 3, different traditional Chinese medicine extracts are mixed and added into 100 mu L of culture solution containing humSCs with good growth state according to the final concentration proportion in the following table, and the growth condition of the human umbilical cord mesenchymal stem cells is observed.
TABLE 1 Final concentration ratio of different Chinese medicinal extract composition
The results are shown in figure 4, the cell growth inhibition effect of the pseudo-ginseng is obvious, and the cell growth rate of the astragalus, the salvia miltiorrhiza and the ginseng is positively correlated with the cell growth rate at the concentration of 0.1-5 mg/ml.
Example 5 establishment of Co-culture System of humSCs and HMC (glomerular cells) and treatment experiment of combination of human umbilical cord mesenchymal Stem cells and Chinese medicinal extract
Making HMC into 1xl06The cell suspension of ml was inoculated into 6-well culture plates, 2ml per well, and after the cells were attached to the 6-well plate and the medium was changed, the upper chamber was placed in the 6-well plate with sterile forceps.
Human umbilical cord mesenchymal stem cells subcultured in example 1 were digested with 0.25% trypsin to prepare a suspension of human umbilical cord mesenchymal stem cells, the cell concentration was adjusted to 1 × 106/ml, the suspension was inoculated into a petri dish soaked in a culture solution in advance, the petri dish was placed in a 37 ℃ and 5% CO2 incubator for CO-culture, cells were starved with a serum-free medium for 24 hours for synchronization, and the cultured human umbilical cord mesenchymal stem cells were combined with group a-H herbal extracts having the concentrations shown in table 1. After 48h of treatment, each group of cells was collected and the cell culture supernatant was collected and stored at-20 ℃.
ELISA detection of FN and IV collagen content in supernatant: and extracting the supernatant of each group, adding the sample to be detected into the corresponding hole of the micropore reaction plate, and adding the biotinylated antibody working solution 50 txl. Shaking and mixing, and incubating at 20-25 deg.C for 120 min. The liquid in the wells was blotted dry, washed thoroughly 5 times with washing solution and dried. Except for blank wells, add enzyme conjugate working solution l00 ul. Shaking and mixing, and incubating at 20-25 deg.C for 30 min. And (4) sucking the liquid in the holes, fully washing the washing liquid for 5 times, and drying. 50ul of each developer A, B is added into each hole, the mixture is gently shaken and evenly mixed, and the mixture is shaded and developed for l0min at the temperature of 37 ℃. After adding 50ul of stop solution, mix by gentle shaking. The absorbance of each well was measured using a microplate reader (450nm wavelength).
The results are shown in fig. 5, which shows that the huMSCs can improve the kidney lesion at the cellular level, show a significant reduction in FN and COL4, and the addition of the traditional Chinese medicine mixture can promote the improvement of the kidney lesion by the huMSCs. Particularly, the experiments prove that the pseudo-ginseng has an inhibition effect on the cell proliferation of humSCs, and the addition of a low-concentration pseudo-ginseng extract (with the final concentration of 10-30ul/ml) can achieve the effect of inhibiting the generation of fibronectin and IV type collagen in the traditional Chinese medicine extract composition.
Experimental example 6 human umbilical cord mesenchymal stem cells combined with various traditional Chinese medicine extracts for treatment
1) Animal feeding
60 male Wistar rats with the cleaning grade and the weight of 200-220 g are provided by national center for sugar engineering technology research of Shandong university.
Feeding all rats in the same room in cages, naturally illuminating, freely taking food and feeding water, wherein the feeding temperature is 18-25 ℃, the relative humidity is 40-50%, and high decibel noise is avoided; ensuring good ventilation for 1 h/time and 3 times/d; the bedding is changed frequently for 2-3 times per week. The experiment is started after the breeding for one week is adapted to the test environment. And (5) fasting for 12 hours before molding without water supply.
2) Preparation of diabetes model
After the rats are adaptively raised for 7 days, the rats are fasted without water prohibition for 12 hours, a single intraperitoneal injection of STZ (50 mg. kg < -1 >) induces diabetes, and the animals in a control group are intraperitoneally injected with an equal volume of 0.1mol/L citrate buffer solution. After STZ 3d injection, blood sugar was measured with a blood glucose meter and urine sugar was measured with urine sugar test paper. Blood sugar is more than or equal to 16.7 mmol.L-1, urine sugar is more than or equal to + + + and the mixture is listed in diabetic animals.
3) Animals were randomized into groups and observed for intervention therapy
The experimental animals are randomly divided into 6 groups, namely a control group, a model group and an A-H traditional Chinese medicine extract combination treatment group.
Wherein the control group is normal rat; the experimental subjects of the model group are the diabetes model rats in the experimental step 2), and are not treated; the experimental object of the MSC treatment group is the diabetes model rat in the experimental step 2), and the human umbilical cord mesenchymal stem cells are treated; the experimental object of the A-I traditional Chinese medicine extract combination treatment group is the diabetes model rat in the experimental step 2), and the human umbilical cord mesenchymal stem cells and various traditional Chinese medicine extracts are subjected to combination treatment;
the administration is performed by intragastric administration to each group of rats every day, and the administration dosage is the mixed solution of the traditional Chinese medicine composition: 10ml/kg, MSC: 2X 106One/only. The Chinese medicinal composition mixed liquor is prepared according to the concentration shown in the table 2.
TABLE 2 concentration configuration of Chinese medicinal materials
4) Glucose tolerance test
After 6 weeks of the experiment, the rats were fasted for 6 hours, and then were gavaged with 20% glucose 1ml 100g-1Body weight (equivalent to 2 g/kg)-1Body weight), the blood glucose levels were measured with a JPS-3+ -type glucometer at fasting (0min) and 30, 60 and 120min after taking sugar, respectively, and the results are shown in table 3 below:
TABLE 3 blood glucose values (mmol. L) of various groups of animals at different time points-1)(x±s)
| Group of | 0min | 30min | 60min | 120min |
| Control | 6.13±1.42 | 8.25±0.97 | 7.54±1.48 | 6.78±1.53 |
| Model (model) | 23.65±3.78 | 34.89±4.85 | 31.11±4.11 | 28.88±3.78 |
| MSC | 22.85±2.58 | 29.58±4.21 | 25.29±3.87 | 22.15±2.85 |
| A | 21.56±0.24 | 25.69±3.51 | 22.54±1.52 | 19.58±3.51 |
| B | 22.38±0.18 | 24.82±2.16 | 19.85±3.08 | 16.56±2.43 |
| C | 19.46±2.82 | 25.66±1.52 | 21.58±3.45 | 18.88±4.22 |
| D | 19.42±0.95 | 23.84±3.56 | 21.48±2.54 | 19.84±1.58 |
| E | 21.84±1.58 | 22.71±2.84 | 20.65±3.88 | 19.33±2.84 |
| F | 20.66±3.21 | 23.14±3.12 | 20.85±2.57 | 18.57±4.52 |
| G | 21.12±2.58 | 23.85±1.56 | 15.85±2.32 | 12.44±3.08 |
| H | 20.84±2.48 | 21.85±3.58 | 13.45±1.58 | 13.59±3.74 |
| I | 18.85±1.59 | 22.11±1.55 | 14.57±1.56 | 13.85±2.08 |
As can be seen from the results in Table 3, the rats in the blank control group had normal glucose-regulating ability. The blood sugar of the rats in the diabetic group is always at a high level 30-60 min after glucose administration, and the blood sugar level is slightly reduced by 120min, which shows that the glucose tolerance of the rats in the diabetic group is obviously reduced and the glucose regulation function is damaged. In the administration group, the effect of reducing blood sugar by using the huMSCs was remarkable, which indicates that the differentiated huMSCs have a potential effect of improving diabetic nephropathy. The A-H combination of the humSCs has better treatment effect, and the data also obviously reflects the remarkable effect of the A-H combination of the traditional Chinese medicine extracts and the humSCs on reducing the blood sugar level. Wherein in groups A-F, radix astragali, Saviae Miltiorrhizae radix or Ginseng radix extract is respectively combined with fructus Schisandrae or Atractylodis rhizoma extract, and Notoginseng radix extract is added to prepare Chinese medicinal extract mixed solution, and when used in combination with huMSCs, the therapeutic effect is better than that of huMSCs alone; the group G-I shows that the best treatment effect is achieved by adding the schisandra chinensis and the bighead atractylodes rhizome extract together, using one of astragalus, salvia miltiorrhiza or ginseng, and using the pseudo-ginseng extract in combination with the humSCs.
According to the detection results, the A-H traditional Chinese medicine extracts and the humSCs can improve the blood sugar regulation and control capability of the injured rats; compared with the traditional Chinese medicine extract and the humSCs which are used independently, the combination of the traditional Chinese medicine extract and the humSCs has better effects of reducing blood sugar and improving renal function; particularly, the low-concentration pseudo-ginseng extract is found to have the effects of obviously improving and assisting huMSCs to slow kidney injury and improving the blood sugar regulation capability in the traditional Chinese medicine extract combination.
While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.
Claims (2)
1. The application of combining umbilical cord mesenchymal stem cells with various traditional Chinese medicine extracts in preparing a medicament for treating hyperglycemia and diabetic nephropathy is characterized in that the various traditional Chinese medicine extracts are a mixture of six traditional Chinese medicine extracts of astragalus, ginseng, salvia miltiorrhiza, bighead atractylodes rhizome, schisandra chinensis and pseudo-ginseng;
the Chinese medicinal extract composition comprises radix astragali, Ginseng radix, and Saviae Miltiorrhizae radix extract, and the total addition amount is 1-10 mg/ml; 10-50ul/ml of bighead atractylodes rhizome and 20-150ul/ml of schisandra extract; and 0.1-30ul/ml of traditional Chinese medicine extract of panax notoginseng;
the extraction method of the traditional Chinese medicine extract comprises the steps of taking ginseng, salvia miltiorrhiza and astragalus, slicing, filtering, drying, grinding, and sieving with a 80-120-mesh sieve to obtain fine powder for later use;
pulverizing fructus Schisandrae chinensis and Notoginseng radix, placing into a container, soaking in 6-16 times of 50-80% ethanol solution, heating and reflux extracting for 3 times, each for 1-6 hr, filtering, mixing filtrates, distilling under reduced pressure to remove ethanol, and concentrating to obtain concentrated solution with relative density of 1.10-1.13 at 60 deg.C;
decocting Atractylodis rhizoma with 12 times, 10 times and 8 times of water for three times, each for 2 hr, mixing the three water extractive solutions, and vacuum concentrating under reduced pressure to 1g medicinal material/ml extractive solution for use.
2. The use as claimed in claim 1, wherein the combination ratio of the multiple Chinese medicinal extracts is 10mg/ml of total extract of radix astragali, Ginseng radix, and Saviae Miltiorrhizae radix; 30ul/ml of bighead atractylodes rhizome and 30ul/ml of schisandra extract; and 10ul/ml of notoginseng Chinese medicine extract.
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