CN112763671A - High-throughput detection method for influence of compound on pancreatic islet function - Google Patents
High-throughput detection method for influence of compound on pancreatic islet function Download PDFInfo
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Abstract
The invention discloses a high-throughput detection method for the influence of a compound on the function of pancreatic islets, which utilizes 1-5 pancreatic islets at least, can screen the action of dozens to hundreds of compounds on glucose-stimulated insulin secretion, and can also be used for detecting the function of the pancreatic islets of different disease animal models, and the method mainly comprises the steps of pancreatic islet culture → pancreatic islet incubation → insulin or glucagon determination. The method has the advantages of low cost, short time consumption and convenient use, and can be applied to drug screening and islet biology research.
Description
Technical Field
The invention relates to a high-throughput detection method for the influence of a compound on pancreatic islet function.
Background
Diabetes is currently one of the most serious health problems worldwide, with about 4.15 million people suffering diabetes and 3.18 million people impaired glucose tolerance among 20-79 years of age worldwide 2015. China is the first major country of diabetics all over the world, the number of patients reaches 1.096 hundred million in 2015, and 130 million people die of diabetes and complications thereof. According to the prediction of the international diabetes union, if no intervention is added, the number of diabetics in the whole world in 2040 years reaches 6.42 hundred million, and the number of patients in China rises to 1.54 hundred million. Diabetes is a disease which is mainly characterized by hyperglycemia due to absolute and relative insufficiency of insulin in a body, and if the hyperglycemia of a patient is not controlled for a long time, the occurrence and development of diabetic cardiomyopathy, nephropathy, retinopathy and neuropathy can be caused, and the health and life of the patient are seriously damaged. Therefore, the pathogenesis and the drug of diabetes are always the key points of research in the medical field, and the promotion of insulin secretion is one of the action mechanisms of the antidiabetic drug.
The pancreatic islets are endocrine tissues of the pancreas, mainly cell clusters consisting of pancreatic islet alpha and beta cells, scattered around the pancreas, and insulin and glucagon produced by the pancreatic islets are main regulating hormones of the body's energy metabolism, especially for the metabolic regulation of carbohydrates, which can cause diabetes if the insulin is not secreted enough for a long time. Current techniques for islet function studies include islet perfusion and islet incubation. The islet perfusion can obtain a dynamic insulin secretion curve, but the islet perfusion is long in time consumption, high in cost and high in technical difficulty. The islet and function research in the traditional method separation culture is not suitable for high-throughput research, and the application of islet function research in the disease mechanism research field and the drug research and development field is limited.
Disclosure of Invention
The invention provides a high-throughput detection method for the influence of a compound on the pancreatic islet function, which can be used for rapidly detecting the influence of the compound on the pancreatic islet function in a large batch.
The purpose of the invention is realized by the following technical scheme:
a method for high throughput detection of the effect of a compound on pancreatic islet function comprising the steps of:
a. culturing the pancreatic islets in an RPMI1640 culture solution for 2-3 days;
b. placing 1-5 pancreatic islets in a 96-well plate, wherein the size error between the pancreatic islets is not more than 20%, adding an incubation liquid, centrifuging, and removing a supernatant;
c. adding an incubation solution into the pancreatic islets, culturing for 30 minutes on a shaking table, centrifuging, and removing a supernatant;
d. the stimulation solution was added to the islets, incubated on a shaker for 30 minutes, centrifuged, the supernatant was collected, and the insulin secretion level in the supernatant was measured.
Preferably, the stimulating solution comprises a control group stimulating solution and a compound group stimulating solution, wherein the control group stimulating solution is formed by adding glucose into the incubation solution, and the compound group stimulating solution is formed by adding glucose with the same concentration and a compound to be tested into the incubation solution; or the control group stimulating solution is obtained by adding leucine and glutamine into the incubation solution, and the compound group stimulating solution is obtained by adding leucine and glutamine with the same concentration and a compound to be detected into the incubation solution;
analyzing whether the compound has the effect of promoting or inhibiting insulin secretion of the insulin by glucose according to the insulin secretion levels cultured by the control group stimulating solution and the compound group stimulating solution, wherein the degree of the promotion or inhibition is; or analyzing whether the compound has the effect of promoting or inhibiting the insulin secretion of the pancreatic islets by leucine and glutamine to the extent of promotion or inhibition;
the concentration of glucose in the stimulation solution is 0-25 mM; or the stimulating solution has leucine concentration of 0-10 mM and glutamine concentration of 2 mM.
Preferably, the concentration of NaCl in the incubation liquid is 115mmol/L, and NaHCO is3Has a concentration of 24mmol/L, a concentration of KCl of 5mmol/L, MgCl2Is 1mmol/L of CaCl2Has a concentration of 2.5 mmol/L, a HEPES concentration of 10mmol/L and a solvent of H2O, adjusting the pH of the solution to 7.4, and adding 0.25% bovine serum albumin.
Preferably, the culture conditions in step a are 37 ℃ and 5% CO2And an incubator with 95% air humidity.
Preferably, the culture conditions in steps c and d are 37 ℃ and 5% CO2A shaking table of an incubator.
The invention also discloses a high-throughput detection method for the influence of the compound on the functions of pancreatic islets (insulin and glucagon secretion), which comprises the following steps:
a. culturing the pancreatic islets in an RPMI1640 culture solution for 2-3 days;
b. placing 15-25 pancreatic islets in a culture device, wherein the size error between the pancreatic islets is not more than 20%, adding an incubation liquid, centrifuging, and removing a supernatant;
c. adding an incubation solution into the pancreatic islets, culturing for 30 minutes on a shaking table, centrifuging, and removing a supernatant;
d. the stimulation solution was added to the islets, incubated on a shaker for 30 minutes, centrifuged, the supernatant was collected, and the insulin and glucagon secretion levels in the supernatant were measured.
Preferably, the stimulating solution comprises a control group stimulating solution and a compound group stimulating solution, wherein the control group stimulating solution is obtained by adding glycine and glucose with different concentrations into an incubation solution, and the compound group stimulating solution is obtained by adding glycine with the same concentration, glucose with different concentrations and a compound to be tested into the incubation solution;
according to the insulin and glucagon secretion levels cultured by the control group stimulating solution and the compound group stimulating solution, the insulin secretion promoting effect of the compound on the pancreatic islets, the promotion or inhibition effect of the glycine on the glucagon secretion stimulating effect and the inhibition effect of the glucose on the glucagon secretion stimulated by the glycine are analyzed, and the promotion or inhibition degree is that, the glucose concentration in the stimulating solution is 0-25 mmol/L, and the glycine concentration is 1 mmol/L.
Preferably, the concentration of NaCl in the incubation liquid is 115mmol/L, and NaHCO is3Has a concentration of 24mmol/L, a concentration of KCl of 5mmol/L, MgCl2Is 1mmol/L of CaCl2Has a concentration of 2.5 mmol/L, a concentration of HEPES of 10mmol/L, and a solvent of H2O, adjusting the pH of the solution to 7.4, and adding 0.25% bovine serum albumin.
Preferably, the culture conditions in step a are 37 ℃ and 5% CO2And an incubator with 95% air humidity.
Preferably, the culture conditions in steps c and d are 37 ℃ and 5% CO2A shaking table of an incubator.
The invention has the following beneficial effects:
the method can measure the influence of the compound on the pancreatic islet secretion level only in about 4 hours (the pancreatic islet experiment time is 1 hour, and the hormone measurement time is 3 hours), the dosage of pancreatic islets is small, and only 1-5 pancreatic islets are needed for measurement. If the culture container selects a 96-well plate, the influence of a plurality of compounds on the islet secretion level can be measured simultaneously, the cost is low, the time consumption is short, the steps are simple, the use is convenient, the result repeatability is good, and the method is widely applied to drug screening and islet biology research.
Drawings
FIG. 1 is a graph showing the effect of an islet function-stimulating drug (GSK-J1500 nmol/L, H3K27 histidine methylene inhibitor) of example 1 on glucose-stimulated insulin secretion, wherein the triangle is the insulin level secretion pattern of islets cultured in control group stimulation solution, and the circle is the insulin level secretion pattern of islets cultured in compound group (GSK-J1);
FIG. 2 is a graph showing the effect of an islet function-stimulating drug (TAK-875) on glucose-stimulated insulin secretion in example 2;
FIG. 3 is a graph showing the effect of the stimulators in example 3 on the secretion of glucagon from mice. The figure shows the secretion of glucagon level in the knockout mouse in a square shape, and the figure shows the secretion of glucagon level in the wild type mouse in a round shape.
Detailed Description
Example 1:
1. islet isolation
Methods for islet isolation are common in the art. The process of isolating the mouse islets is briefly described as follows:
1) mice were anesthetized and fixed to the surgical plate, the abdominal cavity was opened, the bile duct was closed with a hemostat to access the duodenum, and then a 2mg/ml collagenase solution (dissolved in Hanks buffer) was injected into the bile duct using a 5ml syringe and a 31.5 gauge needle until the pancreas was fully filled and stopped.
2) Peeling off pancreas, removing fat and non-pancreatic tissues, transferring the pancreas into a 50ml centrifugal tube, pouring 3ml of 2mg/ml collagenase solution, and shaking in a water bath at 37 ℃ for 4-5 minutes.
3) Immediately after the end of shaking, Hanks buffer was added to 50ml in a 50ml centrifuge tube, centrifuged (2000 rpm, 30 seconds), the supernatant removed, and the remaining tissue pellet was mixed with 5ml Histopaque-1119 solution and shaken well.
4) 5ml of the Histopaque-1077 solution was added slowly and 5ml of Hanks buffer was added slowly and centrifuged (2000 rpm, 10 minutes).
5) Islet tissue is removed from between the stratified fluids, purified islets are picked under a dissecting microscope, washed and cultured.
Islet culture
1) Preparation of RPMI1640 culture solution: 10mmol/L glucose, 2mmol/L glutamine, 10% fetal calf serum, 24mmol/L sodium bicarbonate, 100 units/ml penicillin and 10ug/ml streptomycin are added into the solution, and the pH is adjusted to 7.2.
2) Culturing the separated and purified pancreatic islets in RPMI1640 culture medium at 37 deg.C under 5% CO2And 2-3 days in an incubator with 95% air humidity.
Islet incubation
1) Preparing an incubation liquid Krebs-Ringer bicarbonate buffer (KRBB for short): 115mmol/L NaCl and 24mmol/L NaHCO are added into the solution3, 5 mmol/L KCl, 1 mmol/L MgCl2, 2.5 mmol/L CaCl210mmol/L HEPES, pH was adjusted to 7.4, and 0.25% bovine serum albumin was added.
2) Preparing an irritant liquid: according to the experimental requirements, 5 groups of control group stimulation solutions are prepared by KRBB, wherein the glucose concentration is 0mmol/L, 2mmol/L, 5mmol/L, 10mmol/L and 25mmol/L, and 5 groups of stimulation solutions are prepared and 500nmol/L GSK-J1 is added, and the stimulation solutions are purchased from Sigma company. 3) Selecting islets with the size error not exceeding 20%, placing the islets in a 96-hole flat plate with a V-shaped bottom, placing 1-5 islets in each hole according to requirements, and adding 150ul of KRBB.
4) The plate centrifuge was centrifuged (500 rpm, 20 seconds), the supernatant removed and repeated once more.
5) Adding 150ul KRBB, and standing at 37 deg.C with 5% CO2The supernatant was removed after centrifugation (500 rpm, 20 seconds) by gentle shaking for 30 minutes on a shaker in an incubator.
6) Adding different stimulating solutions, and standing at 37 deg.C and 5% CO2Culture boxThe mixture was shaken for 30 minutes and centrifuged (500 rpm, 20 seconds) to collect the supernatant.
Insulin assay
Taking 10ul of supernatant after the islet stimulation experiment, transferring the supernatant into a 384-empty plate, adding an antibody according to the specification of an HTRF insulin kit, shaking, uniformly mixing, placing at room temperature, incubating for 4 hours, reading by using an HTRF program of a Clariostar microplate reader of BMG, and calculating a hormone secretion value according to a standard curve. The insulin secretion of the supernatant was measured to confirm the effect of the drug on insulin secretion. As can be seen from FIG. 1, the drug was able to improve insulin secretion from the islets of Langerhans of mice stimulated with glucose.
Example 2:
1. islet culture
1) Preparation of RPMI1640 culture solution: 10mmol/L glucose, 2mmol/L glutamine, 10% fetal calf serum, 24mmol/L sodium bicarbonate, 100 units/ml penicillin and 10ug/ml streptomycin are added into the solution, and the pH is adjusted to 7.2.
2) Culturing the separated and purified pancreatic islets in RPMI1640 culture medium at 37 deg.C under 5% CO2And 2-3 days in an incubator with 95% air humidity.
Islet incubation
1) Preparing an incubation liquid Krebs-Ringer bicarbonate buffer (KRBB for short): 115mmol/L NaCl and 24mmol/L NaHCO are added into the solution3, 5 mmol/L KCl, 1 mmol/L MgCl2, 2.5 mmol/L CaCl210mmol/L HEPES, pH was adjusted to 7.4, and 0.25% bovine serum albumin was added.
2) Preparing an irritant liquid: according to the experimental requirements, 2 groups of control stimulating solution with low sugar (3 mmol/L glucose) and high sugar (10 mmol/L glucose) are prepared by KRBB, and different compounds (an example compound is TAK-875 with the concentration of 1 umol/L; and a positive control is Exendin-4 with the concentration of 100 nmol/L) are additionally added into the stimulating solution which is prepared into 2 groups and is the same as the control group.
3) Selecting islets with the size error not exceeding 20%, placing the islets in a 96-hole flat plate with a V-shaped bottom, placing 1-5 islets in each hole according to requirements, and adding 150ul of KRBB.
4) The plate centrifuge was centrifuged (500 rpm, 20 seconds), the supernatant removed and repeated once more.
5) Adding 150ul KRBB, and standing at 37 deg.C with 5% CO2The supernatant was removed after centrifugation (500 rpm, 20 seconds) by gentle shaking for 30 minutes on a shaker in an incubator.
6) Adding different stimulating solutions, and standing at 37 deg.C and 5% CO2The supernatant was collected by centrifugation (500 rpm, 20 seconds) with gentle shaking on a shaker in an incubator for 30 minutes.
Insulin assay
Taking 10ul of supernatant after the islet stimulation experiment, transferring the supernatant into a 384-empty plate, adding an antibody according to the specification of an HTRF insulin kit, shaking, uniformly mixing, placing at room temperature, incubating for 2-4 hours, reading by using an HTRF program of a Clariostar microplate reader of BMG, and calculating a hormone secretion value according to a standard curve. The insulin secretion of the supernatant was measured to confirm the effect of the drug on insulin secretion. As can be seen from FIG. 2, 1) the 10mmol/L glucose-stimulated insulin secretion is significantly higher than the stimulation capacity of 3mmol/L glucose; 2) the 100nmol/L Exendin-4 obviously improves the insulin secretion stimulated by 10mmol/L glucose; 3) the compound TAK-875 stimulates insulin secretion stimulated by 10mmol/L glucose. It can be seen that the compound TAK-875 is capable of improving the secretion of glucose and insulin, and is suitable for the development of drugs for diabetes.
Example 3:
1. islet culture
1) Preparation of RPMI1640 culture solution: 10mmol/L glucose, 2mmol/L glutamine, 10% fetal calf serum, 24mmol/L sodium bicarbonate, 100 units/ml penicillin and 10ug/ml streptomycin are added into the solution, and the pH is adjusted to 7.2.
2) Culturing pancreas islet of knockout mouse and pancreas islet of wild mouse at 37 deg.C under 5% CO with RPMI1640 culture medium2And 2-3 days in an incubator with 95% air humidity.
Islet incubation
1) Preparing an incubation liquid Krebs-Ringer bicarbonate buffer (KRBB for short): 115mmol/L NaCl and 24mmol/L NaHCO are added into the solution3, 5 mmol/L KCl, 1 mmol/L MgCl2, 2.5 mmol/L CaCl210mmol/L HEPES, pH was adjusted to 7.4, and 0.25% bovine serum albumin was added.
2) Preparing an irritant liquid: according to the experimental requirements, 5 groups of control groups of stimulating solution are prepared by KRBB, wherein the concentration of glycine is 1mmol/L, and the concentration of glucose is 0mmol/L, 5mmol/L, 100 mmol/L and 20 mmol/L.
3) Selecting islets with the size error not exceeding 20 percent, placing the islets in a V-shaped bottom 96-hole plate, and adding 150ul of KRBB to 20 islets in each hole according to the experiment requirements.
4) The plate centrifuge was centrifuged (500 rpm, 20 seconds), the supernatant removed and repeated once more.
5) Adding 150ul KRBB, and standing at 37 deg.C with 5% CO2The supernatant was removed after centrifugation (500 rpm, 20 seconds) by gentle shaking for 30 minutes on a shaker in an incubator.
6) Adding different stimulating solutions, and standing at 37 deg.C and 5% CO2The supernatant was collected by centrifugation (500 rpm, 20 seconds) on a shaker in an incubator for 30 minutes and stored in a freezer at-20 ℃ until hormone measurement.
Insulin and glucagon assay
Taking 10ul of supernatant after an islet stimulation experiment, transferring the supernatant into a 384-empty plate, adding an antibody according to the instruction of an insulin kit or a glucagon determination kit, shaking, uniformly mixing, incubating at room temperature for 2-4 hours, reading by using a Clariostar microplate reader HTRF program of BMG, calculating a hormone secretion value according to a standard curve, and determining the influence of glucose and glycine on the secretion of mouse insulin and glucagon by determining the simultaneous secretion of the insulin and the glucagon in the supernatant. As can be seen from FIG. 3, wild-type mouse islets were sensitive to glycine stimulation and also sensitive to glucose-inhibited glucagon secretion. However, pancreatic islets of knockout mice are abnormal in both glycine-stimulated glucagon secretion and glucose-inhibited glucagon.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A method for high throughput detection of the effect of a compound on pancreatic islet function comprising the steps of:
a. culturing the pancreatic islets in an RPMI1640 culture solution for 2-3 days;
b. placing 1-5 islets in a perforated plate, wherein the size error between the islets is not more than 20%, adding an incubation liquid, centrifuging, and removing a supernatant; the process is repeated again;
c. adding an incubation solution into the pancreatic islets, incubating for 30 minutes on a shaking table, centrifuging, and removing a supernatant;
d. adding a stimulating solution into the pancreatic islets, incubating for 30 minutes on a shaking table, centrifuging, collecting supernatant, measuring the insulin secretion level in the supernatant, and analyzing the influence of the compound to be tested on the insulin secretion according to the insulin secretion level.
2. A method for high throughput detection of the effect of compounds according to claim 1 on pancreatic islet function, characterized in that: the stimulating solution comprises a control group stimulating solution and a compound group stimulating solution, wherein the control group stimulating solution is formed by adding glucose with a designed concentration into an incubation solution, and the compound group stimulating solution is formed by adding glucose with the same concentration and a compound to be tested with the designed concentration into the incubation solution; or adding leucine and glutamine into the incubation liquid to form a control group stimulation liquid, adding leucine and glutamine with the same concentration and a to-be-detected compound with the designed concentration into the incubation liquid to form a compound group stimulation liquid, wherein the concentration of glucose in the stimulation liquid is 0-25 mmol/L; or the concentration of leucine in the stimulation solution is 0-10 mmol/L, and the concentration of glutamine is 2 mmol/L.
3. A method for high throughput detection of the effect of a compound according to claim 1 or 2 on pancreatic islet function, characterized in that: the concentration of NaCl in the incubation liquid is 115mmol/L, and NaHCO is3Has a concentration of 24mM, a concentration of KCl of 5mmol/L, MgCl2Is 1mmol/L of CaCl2Has a concentration of 2.5 mmol/L, a HEPES concentration of 10mmol/L and a solvent of H2O, adjusting the pH value of the solution to 7.4, and then adding 0.25 percent of bovine serum albuminWhite.
4. A method for high throughput detection of the effect of a compound according to claim 3 on pancreatic islet function, wherein: the culture conditions in step a were 37 ℃ and 5% CO2And a shaker incubator with 95% air humidity.
5. A method for high throughput detection of the effect of compounds according to claim 4 on pancreatic islet function, characterized in that: the culture conditions in steps c and d were 37 ℃ and 5% CO2The shaking incubator.
6. A method for high throughput detection of the effect of a compound on pancreatic islet function comprising the steps of:
a. culturing the pancreatic islets in an RPMI1640 culture solution for 2-3 days;
b. placing 15-25 pancreatic islets in a porous plate, wherein the size error between the pancreatic islets is not more than 20%, adding an incubation liquid, centrifuging, removing a supernatant, and repeating the step once;
c. adding an incubation solution into the pancreatic islets, culturing for 30 minutes on a shaking table, centrifuging, and removing a supernatant;
d. adding stimulating solution into pancreatic islet, culturing for 30 min on shaking table, centrifuging, collecting supernatant, measuring secretion levels of insulin and glucagon in the supernatant, and analyzing influence of the compound to be detected on secretion of insulin and glucagon according to the secretion levels of insulin and glucagon.
7. A method for high throughput detection of the effect of compounds according to claim 6 on pancreatic islet function, characterized in that: the stimulating solution comprises a control group stimulating solution and a compound group stimulating solution, wherein the control group stimulating solution is formed by adding glycine and glucose with designed concentration into an incubation solution, the compound group stimulating solution is formed by adding glycine, glucose and a compound to be tested with the same concentration into the incubation solution, the concentration of the glucose in the stimulating solution is 0-25 mmol/L, and the concentration of the glycine is 1 mmol/L.
8.High throughput method for the detection of the effect of compounds according to claim 6 or 7 on pancreatic islet function, characterized in that: the concentration of NaCl in the incubation liquid is 115mmol/L, and NaHCO is3Has a concentration of 24mmol/L, a concentration of KCl of 5mmol/L, MgCl2Is 1mmol/L of CaCl2Has a concentration of 2.5 mmol/L, a HEPES concentration of 10mmol/L and a solvent of H2O, adjusting the pH of the solution to 7.4, and adding 0.25% bovine serum albumin.
9. A method for high throughput detection of the effect of a compound according to claim 8 on pancreatic islet function, wherein: the culture conditions in step a were 37 ℃ and 5% CO2And a shaker incubator with 95% air humidity.
10. A method for high throughput detection of the effect of a compound according to claim 9 on pancreatic islet function, characterized in that: the culture conditions in steps c and d were 37 ℃ and 5% CO2The shaking incubator.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000078724A2 (en) * | 1999-06-18 | 2000-12-28 | Eli Lilly And Company | Imidazolinderivatives their preparation and their pharmaceutical use |
| CN1844925A (en) * | 2006-04-24 | 2006-10-11 | 东莞市博康健医药科技有限公司 | Method for determining bioactivity of recombinant insulin secretagogue and application thereof |
| CN103154240A (en) * | 2010-08-09 | 2013-06-12 | 武田药品工业株式会社 | Method of producing pancreatic hormone-producing cells |
| CN105368771A (en) * | 2015-12-04 | 2016-03-02 | 东南大学 | Mouse islet separation and purification method |
| CN108992442A (en) * | 2018-07-12 | 2018-12-14 | 上海交通大学医学院附属瑞金医院 | Application of MS-275 in preparation of medicine for promoting insulin secretion |
-
2019
- 2019-11-04 CN CN201911063732.4A patent/CN112763671A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000078724A2 (en) * | 1999-06-18 | 2000-12-28 | Eli Lilly And Company | Imidazolinderivatives their preparation and their pharmaceutical use |
| CN1844925A (en) * | 2006-04-24 | 2006-10-11 | 东莞市博康健医药科技有限公司 | Method for determining bioactivity of recombinant insulin secretagogue and application thereof |
| CN103154240A (en) * | 2010-08-09 | 2013-06-12 | 武田药品工业株式会社 | Method of producing pancreatic hormone-producing cells |
| CN105368771A (en) * | 2015-12-04 | 2016-03-02 | 东南大学 | Mouse islet separation and purification method |
| CN108992442A (en) * | 2018-07-12 | 2018-12-14 | 上海交通大学医学院附属瑞金医院 | Application of MS-275 in preparation of medicine for promoting insulin secretion |
Non-Patent Citations (2)
| Title |
|---|
| 刘振平 等: "不同氨基酸对克隆的胰岛β 细胞株INS-1E细胞作用的比较", 《氨基酸和生物资源》 * |
| 郭阳艳 等: "黄芩素对大鼠胰岛素分泌的作用及机制研究", 《中国药理学通报》 * |
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