Patent Application
20180161375
The present invention is related to a therapeutic method for repairing pancreas injuries, particularly with providing a plurality of pretreated adipose-derived stem cells (ADSC) and epigallocatechin gallate (EGCG) of green tea. And EGCG of green tea can enhance the ability of the adipose-derived stem cells for repairing damaged tissue.
The clinical determination of diabetes is that the fasting blood-glucose value is higher than 110 mg/dl and the glycated hemoglobin HbA1c value is higher than 6.5%. The cause of hyperglycemia is something wrong in the metabolic mechanism of blood glucose. The insulin secreted from pancreas is the foremost protein for metabolizing blood glucose, so the hyperglycemia is caused by abnormal insulin secretion.
In terms of Type I diabetes, the secretory volume of insulin is insufficient, a small amount of insulin cannot metabolize a large amount of blood glucose, high-concentration glucose accumulates in blood, forming hyperglycemia. Since the primary cause of Type I diabetes is hypoinsulinism, the Type I diabetes is mostly caused by dysfunction of pancreas. There are many factors in forming Type I diabetes, such as pancreatic cell death caused by autoimmune disease, pancreas hypofunction resulted from heredity problems or pancreas function damaged by virus infection, these factors can fail the pancreas function and cause insufficient secretory volume of insulin, the β cell of pancreas is the insulin producer in hysinsulinism. Previous findings on mice showed that if the β cells of injured pancreas were repaired, the volume of insulin increased relatively, and the blood-sugar content returned to normal value.
In terms of the management of Type I diabetes at present, besides insulin injection and pancreas transplantation, there are control of habits and customs (e.g. regular exercise, low-calorie low-oil diet, body weight control) and pharmacotherapy. Besides the aforesaid methods, some documents mentioned transplanting stem cells to treat diabetes. However, treating diabetes by transplanting stem cells is mostly seen in fundamental medical research reports, and there are few documents about clinical treatment of diabetes by transplanting stem cells.
In addition, it is indicated that a high glucose environment injures stem cells, degrading the mobility or restorability of cells, even increasing the oxidizing pressure in stem cells. In other words, how to let the stem cells exist in an inappropriate environment, such as a high glucose environment, and repair the damaged organs effectively, such as pancreas, is an important topic.
Hence, how to let the stem cells exist in unfavorable environments for cell growth, such as high glucose and high oxidizing pressure microenvironments; to further improve and treat the damaged organs of organisms, and to solve the deficiencies in the known techniques are urgent issues for related technical fields to solve.
Accordingly, the present invention provides the method for repairing a damaged pancreas in a subject, wherein the method comprising administrating to said subject a plurality of pretreated adipose-derived stem cells and an ester type catechin, wherein the plurality of pretreated adipose-derived stem cells are cultured with the ester type catechin.
Preferably, the damaged pancreas causes diabetes or hyperglycemia.
Preferably, the damaged pancreas is a plurality of damaged beta cells.
Preferably, the pretreated adipose-derived stem cells are pretreated with the ester type catechin for 2 hours.
Preferably, the concentration of ester type catechin for the pretreated adipose-derived stem cells is 5-25 μM.
Preferably, the concentration of ester type catechin for the pretreated adipose-derived stem cells is lower than 20 μM.
Preferably, the pretreated adipose-derived stem cells have higher viability in a high glucose environment, wherein the high glucose environment means the glucose concentration is 22 mM-44 mM, mainly 33 mM, the high viability is 80%˜100%.
Preferably, the pretreated adipose-derived stem cells increase expression level of a group of survival related protein in the damaged pancreas, wherein the group of survival related protein comprising p-IGF1R, PI3K, p-PI3K, Akt, p-Akt, p-Bad and Bcl-xL, the percentage of increase is 50%˜100%.
Preferably, the pretreated adipose-derived stem cells increase expression level of a group of Sirt1 pathway related protein in the damaged pancreas, wherein the group of Sirt1 pathway related protein comprising p-AMPK and Sirt1, the percentage of increase is 50%˜100%.
Preferably, the pretreated adipose-derived stem cells reduce expression level of a group of cell apoptosis pathway related protein in the damaged pancreas, wherein the group of cell apoptosis pathway related proteins comprising FADD, Caspase 8, Bax, Cytochrome C and Caspase 3, the percentage of decrease is 40%˜80%.
Preferably, the pretreated adipose-derived stem cells reduce expression level of a fibrosis related protein in the damaged pancreas, wherein the fibrosis related protein is TGF-β, the percentage of decrease is 40%˜80%.
Although the present invention has been described in terms of specific exemplary embodiments and examples, it will be appreciated that the embodiments disclosed herein are for illustrative purposes only and various modifications and alterations might be made by those skilled in the art without departing from the spirit and scope of the invention as set forth in the following claims.
Although the present invention has been described in terms of specific exemplary embodiments and examples, it will be appreciated that the embodiments disclosed herein are for illustrative purposes only and various modifications and alterations might be made by those skilled in the art without departing from the spirit and scope of the invention as set forth in the following claims.
The present invention provides a therapeutic method for treating pancreas injuries. The therapeutic method administrating to said subject a plurality of pretreated adipose-derived stem cells and an ester type catechin.
The following embodiments are provided for illustrative description, not to limit the scope of the present invention.
1. Animal Experiment Design
The 8 months old Wistar strain male rats (bought from Lasco company) are divided into four groups, which are normal group (Sham), STZ (55 mg/kg) induced diabetes group (DM), diabetes+autogenous ADSC treatment group (DM+ADSC) and diabetes+green tea EGCG pretreated autogenous ADSC treatment group (DM+E-ADSC), as shown in Table 1. The rats are bred in the animal house for 12 hours during the day and 12 hours at night circularly. The food and water are taken freely during breeding. Two rats live in one animal cage, the animal litter is changed every two days during breeding. When the blood glucose of the rats of diabetes groups increase to 200 mg/dl, the diabetes is determined. The determined diabetes groups are treated by transplanting autogenous stem cells one month later. In terms of feedback of autogenous stem cells, 1×105˜1×107 stem cells are fed back into each rat through vena caudalis.
2. Blood Glucose Analysis
For the rat blood glucose test, the fasting caudal vein blood is tested by Roche blood glucose meter (Accu-Chek Active).
1. ADSC Extraction
The adipose tissue in the abdominal cavity of an 8 months old Wistar strain male rat is extracted surgically. The adipose tissue is cut in appropriate size, cleaned with physiological saline solution containing antibiotics, the cleaned adipose tissue is put in the physiological saline solution containing Type II collagenase (0.01%), heated and stirred in 37° C. water bath for about 1 hour, centrifugated at 3000 rpm at room temperature for about 10 minutes, the lower precipitate is extracted and put in the cell culture dish for cell culture.
2. Stem Cell Characteristic Analysis
Before the autotransplantation of stem cells for treatment, it is required to confirm whether the transplanted cells are stem cells or not. There are two kinds of analysis mode to determine stem cells. The first mode uses flow cytometer to analyze the positive marker and negative marker on the stem cell membrane. The characteristic of stem cells is that the higher the expression level of positive marker is, the lower is the expression level of negative marker. The other mode is differentiation experiment, to observe whether the stem cells are able to be differentiated into other heterogeneous cells. According to
The cells are cultured in 24-well dish, the cells are medicated, the culture medium is removed, washed with PBS buffer (three times), cultured in culture medium containing 0.5 mg/ml MTT for 3˜4 hours, the culture medium is removed, washed with PBS buffer, mixed with 1 ml isopropanol to dissolve purple formazan crystal, the O.D.570 nm absorbance value determination is implemented 5 minutes later.
The cell viability of stem cells under the stimulation of high glucose environment is shown in
The protein is quantified by Bradford protein assay. The principle is that the protein and Coomassie brilliant blue G-250 can form a blue compound. The darker the blue is, the higher is the protein content. In terms of the test method, a series of BSA at given concentration is mixed with 1/5 Bradford protein dye by volume, a standard curve is made from the brightness of 595 nm visible light, and the O.D. value of sample is measured in the same way, the concentration of sample protein can be obtained according to the standard curve.
For the rat blood glucose test, the fasting caudal vein blood is tested by Roche blood glucose meter (Accu-Chek Active).
Changes in blood glucose values of various groups of experimental animals before and after stem cell treatment
An amount of 40 ug protein sample is mixed with PBS solution and 5× loading dye uniformly and boiled for 10 minutes before SDS-polyacrylamide slab gel electrophoresis analysis. The upper colloid of SDS-polyacrylamide slab gel electrophoresis is 3.75% Stacking gel, the lower colloid is 5% and 12% Separating gel. The prepared slab gel is fixed to the electrophoretic apparatus, and the electrophoresis tank is filled up with electrophoretic buffer (Electrode buffer). Afterwards, the treated protein sample solution is poured into the U-shaped channel formed in the slab gel, electrophoresed with 75V. The protein transfer is implemented after electrophoresis, the colloid is extracted, the colloid is laid on a wetted Whatman 3M filter paper. The colloid is covered with a methanol wetted PVDF membrane, and covered with a wetted 3M filter paper, and it is put in Transfer Holder after the intermediate bubbles are swept slightly by a glass rod, and then it is put in Electrotransfer Tank (containing Transfer buffer) at 4° C., after 1-hour transfer under 100 V, the PVDF membrane is taken out and immersed in 5% (w/v) skimmed milk (Blocking buffer)(PBS-non-fat milk powder) and shaken at room temperature for one hour. The PVDF membrane is put in a 4° C. refrigerator, reacting with primary antibody overnight, and then cleaned with washing buffer twice, 10 minutes each time. It is dumped after the last cleaning. It reacts with horseradish peroxidase conjugated secondary antibody for 2 hours, the PVDF membrane is cleaned in the same way. Finally, the PVDF membrane is immersed in 4 ml substrate buffer for color reaction.
According to
According to
The pancreatic tissue is taken from the sacrificed rat, soaked in formalin to fix the tissue, and then dehydrated by ethanol, the pancreatic tissue is embedded in paraffin and cut up into 0.2 μm tissue slice. In HE staining, the tissue slice is dewaxed and fixed by ethanol and formalin, stained by hematoxylin and eosin for observation. In Masson's Trichrome staining, the tissue slice is dewaxed and fixed by ethanol and formalin, and stained by the following stains, Bouin's stain 15 minutes, Weiger stain 10 minutes, Beibrich stain 5 minutes, PPA stain 10 minutes and Aniline blue stain 5 minutes.
1. Experimental Animal Pancreatic Tissue Slice Analysis
2. Analysis of Experimental Animal Pancreatic Tissue Slice Fibrosis
3. Statistical Analysis
The analysis results of each specimen/sample are obtained by analyzing the data of three or more experiments. The ANOVA is used for statistics, p<0.05 represents significant difference.
According to the experimental results, the stem cell treatment can reduce the blood glucose of the rat with diabetes, the mechanism is to scale up the pancreatic tissue survival related protein, scale up the Sirt1 pathway related protein, scale down the expression level of apoptosis pathway related protein, the size of insular tissue of pancreas is restored, so as to restore the pancreas function, and the blood glucose value is scaled down. Meanwhile the stem cell treatment can scale down the fibrosis related protein expression in pancreatic tissue, so that the pancreatic tissue fibrosis is degraded, the pancreas function is restored indirectly.
The green tea EGCG pretreated stem cells (DM+E-ADSC) protect the pancreatic tissue better than the untreated stem cells (DM+ADSC). It is found in the cell experiment that the survival related protein p-Akt of stem cells is scaled down in high glucose environment, so that the viability of stem cells in high glucose environment decreases. The p-Akt of stem cell is scaled up after the green tea EGCG pretreatment, so that the cell viability of stem cells increases in high glucose environment. According to the experimental results, when the stem cells are transplanted to the diabetic animal, the high glucose environment resulted from diabetes scales down the stem cell survival p-Akt, so that the viability of stem cells in the diabetic animal body is reduced, and the treatment effect is degraded. If the stem cells are pretreated with EGCG before the transplantation to the diabetic animal for treatment, the p-Akt survival is scaled up, so that the resistance of stem cells to the high glucose environment caused by diabetes is enhanced (EGCG pretreated stem cells are unlikely to die in high glucose environment), and then there are considerable stem cells survive the diabetic animal body, certainly the function to protect (repair) pancreatic tissue is better.
The following key points are derived from experimental conclusions: (1) the EGCG pretreatment can scale up the resistance of stem cells to high glucose environment; (2) the EGCG treated stem cells have better abilities to repair the damaged pancreatic tissue and to reduce blood glucose than the stem cells without any treatment; (3) the EGCG treated stem cells repair the damaged pancreatic tissue by scaling up the survival related protein in pancreatic tissue, scaling up the Sirt1 pathway related protein, scaling down the apoptosis related protein and scaling down the fibrosis related protein.
Although the present invention has been described in terms of specific exemplary embodiments and examples, it will be appreciated that the embodiments disclosed herein are for illustrative purposes only and various modifications and alterations might be made by those skilled in the art without departing from the spirit and scope of the invention as set forth in the following claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 106138102 | Nov 2017 | TW | national |
This application is a Continuation-in-Part of co-pending application Ser. No. 15/160,519, filed on May 20, 2016, for which priority is claimed under 35 U.S.C. § 120; and this application claims priority of Application No. 106138102 filed in Taiwan on Nov. 3, 2017 under 35 U.S.C. § 119; the entire contents of all of which are hereby incorporated by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15160519 | May 2016 | US |
| Child | 15889703 | US |
