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CN109576225A - Inhibit the method for apoptosis of mesenchymal stem cell using Rspo1 - Google Patents

Inhibit the method for apoptosis of mesenchymal stem cell using Rspo1 Download PDF

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CN109576225A
CN109576225A CN201811576967.9A CN201811576967A CN109576225A CN 109576225 A CN109576225 A CN 109576225A CN 201811576967 A CN201811576967 A CN 201811576967A CN 109576225 A CN109576225 A CN 109576225A
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mesenchymal stem
stem cell
rspo1
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解军
程肖霞
杨丽红
杨巧艳
张慧超
谭奎
张娟
刘丹
李仁科
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Shanxi Medical University
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Abstract

The invention discloses a kind of utilizationsRspo1The method for inhibiting apoptosis of mesenchymal stem cell is to carryRspo1The Adenovirus Transfection rat bone marrow mesenchymal stem cells of gene obtain stable expression after transfecting 72hRspo1Mesenchymal stem cell strain.It is detected through flow cytometer, immunofluorescence and qPCR,Rspo1Transfection enters mesenchymal stem cell.Fluidic cell is carried out to mesenchymal stem cell after transfection and the bis- dyes of AO-EB detect Apoptosis situation, it was demonstrated thatRspo1By inhibiting apoptosis to improve the survival condition of mesenchymal stem cell, condition is provided for the external fast-growth of mesenchymal stem cell.

Description

It utilizesRspo1Inhibit the method for apoptosis of mesenchymal stem cell
Technical field
The present invention relates to a kind of methods for inhibiting apoptosis of mesenchymal stem cell, utilize adenovirus more particularly to a kind of Mediate geneRspo1After gene-transfected BMSCs, inhibit the method for apoptosis of mesenchymal stem cell.
Background technique
Mesenchymal stem cell (Bone marrow mesenchymal stem cells, BMSCs) be easy to get and Mass propagation can be cultivated in vitro, there is multinomial differentiation potential, be not related to ethics problem, be the ideal choosing of stem-cell therapy disease It selects.There is research report to claim, experienced the acute myocardial infarction AMI of percutaneous coronary intervention (pci) and cardioprotective treatment at 96 In patient, the effect that autologous bone marrow mesenchymal stem cells transplanting improves heart can continue to after operation 6 months.Recently, preclinical Experimental study shows that genetic strategies may play a crucial role in the existence and differentiation for improving mescenchymal stem cell.
BMSCs has been achieved for effective therapeutic effect in cellular transplantation therapy, and becoming in regenerative medicine field can have Imitate the seed cell to play a role.However, transplanting intracorporal BMSCs survival rate and vigor is limited, quality of life of patients is mentioned It is high not satisfactory.
Preclinical phase experimental study shows that genetic strategies may play in the existence and differentiation for improving mescenchymal stem cell Key effect.Therefore, effect of the mesenchymal stem cell in disease treatment is improved extremely using a gene or one group of gene It closes important.
Shu-Li Zhao etc. is studies have shown that be overexpressed geneMKMesenchymal stem cell can reduce tire mouse heart source The apoptosis rate of cell line H9C2 cell line, and then the survival rate of cell is improved, and compared with mesenchymal stem cell is applied alone, change The effect of kind myocardial infarction Cardiac Function is more obvious.mirco RNA-383Can be enhanced glial cell line-derived neurotrophic because The expression of son, mescenchymal stem cell is passed throughmirco RNA-383Gene modification is repaired through can promote the gene that correlation factor is expressed Decorations, can be improved mescenchymal stem cell to the therapeutic effect of spinal cord injury.BMSCs withBMP-2The BMSCs of transfection can improve bone The gastrocnemius tendon biological function of marrow intraluminal grafting, andBMP-2The BMSCs of transfection then more significantly promotes tendon-bone healing.
Rspo1It is a member of Rspo family.The family regulates and controls the growth and development process of animal, including blood vessel, muscle, bone The formation of equal tissues and animal limb, reproduction, digestion and respiratory system development, at the same also with the proliferation of various kinds of cell with point Change process is closely related.Rspo1 is a kind of secretory protein that molecular weight is 35KDa, can contain weight in conjunction with rich in leucine G-protein coupling receptor (LGR) 4-6 of complex sequences, and phosphorylation can occur with soluble Wnt3a co-induction LRP6, moreover it is possible to Promote cytoplasm stable and β-catenin is accumulated in nucleus.These protein conformation varying effects for cell proliferation, differentiation It plays an important role with the maintenance of cell stemness.Reparation of the Rspo1 after the proliferation of cell and differentiation, the decision of gender, damage, individual Growth course all play important biological action.
Therefore, by BMSCs withRspo1It is combined, to improve the survival condition of BMSCs, thus for the external fast of BMSCs Speed amplification provides new experiment and theoretical foundation, and its application is very necessary with disease treatment.
Summary of the invention
The object of the present invention is to provide a kind of utilizationsRspo1Inhibit the method for apoptosis of mesenchymal stem cell.
The research of the invention finds that culture rat bone marrow mesenchymal stem cells are extracted, it willRspo1Pass through adenovirus transfection Into in mesenchymal stem cell, it is able to suppress the apoptosis rate of rat bone marrow mesenchymal stem cells.Rspo1 has inhibition rat bone The latent effect of bone marrow-drived mesenchymal stem apoptosis.
Based on this, the invention proposes a kind of utilizationsRspo1Inhibit the method for apoptosis of mesenchymal stem cell, the side Method is to carryRspo1The adenovirus transfection mesenchymal stem cell of gene, makesRspo1It is dry thin into medulla mesenchyma In born of the same parents, culture obtains rotaring redyeing gene in culture medium without double antibodyRspo1Mesenchymal stem cell.
In the above method of the present invention, the mesenchymal stem cell for being transfected is that in vitro medulla mesenchyma is dry thin Born of the same parents.
Further, the mesenchymal stem cell is isolated rat mesenchymal stem cell.
In turn, present invention preferably uses the isolated rat mesenchymal stem cells of culture to P3 to be transfected.
Cell suspension is made in the mesenchymal stem cell by the present invention, in 37 DEG C, 5% CO2It is cultivated for 24 hours in incubator Afterwards, Adenovirus Transfection is carried out with MOI=100.
In the above method of the present invention, preferably by the carryingRspo1The adenovirus vector of gene be configured to concentration 2 × 108The adenopathy venom of PFU/ml is used for gene-transfected BMSCs.
The present invention is with adenovirus mediatedRspo1The transfection time of gene-transfected BMSCs is 72h.
The present invention is using adenovirus as transfection carrier.Adenovirus (AdV) belongs to the DNA of the non-development of Adenoviridae icosahedron Virus is a kind of virus with 34~43kb of double-strand genome, diameter about 90nm.AdV mainly in combination with Coxsack adenovirus by Body (CAR) enters cell by clathrin-mediated endocytosis effect.To be contaminated independently of host after AdV genome infection cell The form of colour solid exists, therefore host cell BMSCs gene is hardly interfered in the expression of AdV genomeRspo1 Normal table It reaches.
The present invention is in turn to above-mentioned adenovirus mediatedRspo1Obtained mesenchymal stem cell is transfected to be detected, with VerifyingRspo1To the inhibiting effect of apoptosis of mesenchymal stem cell, carries out research improvement mesenchymal stem cell and give birth in vitro The method for depositing state.
By rightRspo1The mesenchymal stem cell of transfection carries out flow cytometer and the bis- dye detections of AO-EB, and detection is dry The apoptosis situation of cell, discoveryRspo1The apoptosis rate for transfecting obtained mesenchymal stem cell can be significantly suppressed.
Rspo1It is able to suppress the apoptosis of mesenchymal stem cell, improves the survival condition of mesenchymal stem cell, is The in vitro culture of mesenchymal stem cell provides new experiment and theoretical foundation, provides condition for stem-cell therapy disease.
Detailed description of the invention
Fig. 1 is culture gained respectively for the cellular morphology figure of BMSCs.
Fig. 2 is the skeletonization of BMSCs, adipogenic induction differentiated result figure.
Fig. 3 is the streaming qualification result figure of BMSCs specific surfaces molecule.
Fig. 4 is the transfection efficiency figure of fluorescence microscope detection Adenovirus Transfection BMSCs.
Fig. 5 is the transfection efficiency figure of flow cytometer detection Adenovirus Transfection BMSCs.
Fig. 6 is in qPCR detection BMSCsRspo1Expression quantity.
Fig. 7 is the apoptosis situation of AnnexinV-APC/7-AAD flow cytometer detection BMSCs.
Fig. 8 is the apoptosis situation of the bis- dye detection BMSCs of AO-EB.
Fig. 9 is apoptosis-related protein Bax, Caspase3 and Cleavage- in Western blot detection BMSCs The expression quantity of Caspase3.
Specific embodiment
Following embodiments are only the preferred technical solution of the present invention, are not used to carry out any restrictions to the present invention.For For those skilled in the art, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made Any modification, equivalent substitution, improvement and etc., should all be included in the protection scope of the present invention.
Embodiment 1: the extraction of rat bone marrow mesenchymal stem cells.
The SD rat cervical dislocation of 80~100g is put to death, dorsal position is fixed on aseptic operation table, and cotton balls dips Iodophor Double hind legs and entire abdomen are sterilized, is sterilized 3 times repeatedly.Double hind leg skins are cut off with sterile scissors, blunt separation skin, exposure Musculature out shears off double hind legs to femoral head, is immersed in 75% alcohol and sterilizes 5min.
Double hind legs are transferred to superclean bench, pick all musculatures and fibula with tweezers and scissors, only surplus femur And shin bone.Femur and shin bone are soaked in PBS solution, bone both ends are cut with scissors, expose myeloid tissue.Use 1ml Syringe draw BMSCs culture medium, repeatedly suction irrigation ossis for several times, until ossis from red become white, obtain bone Marrow suspension.
The marrow suspension of acquisition is moved into culture bottle, 37 DEG C, 5% CO2It is cultivated in incubator.Conventionally will The BMSCs cell of culture is passed on, until passing to the 3rd generation cell (P3).
Cultivate cellular morphology such as Fig. 1 of each generation BMSCs.P0 is smaller for cell volume, and there is protrusion stretching at edge, and form is more Kind multiplicity, there is polygonal, spindle shape, irregular shape (Figure 1A);P1 increases for cell volume, in irregular shape and have more multiple Folded, disorganized, there is more bright round hematopoietic cell (Figure 1B) on surface;P2 start for the cell arrangement of cell it is regular, The bright round hematopoietic cells number in surface reduces (Fig. 1 C);P3 for cell be in spindle shape, size shape is uniform, marshalling and Direction is more consistent (Fig. 1 D).Cytomorphology shows that cultivated cell has form similar with BMSCs.
Embodiment 2: the osteogenic adipogenesis induction of rat bone marrow mesenchymal stem cells breaks up identification.
The P3 cell of 1 degrees of fusion 90% or so of Example prepares cell suspension.By cell suspension according to 1 × 105 A/ml is inoculated in 6 orifice plates, and 2ml BMSCs culture medium, 37 DEG C, 5% CO is added in every hole2It cultivates in incubator, carries out into respectively Bone and adipogenic induction differentiation.
When cell fusion degree reaches 60~70%, culture medium is discarded, every hole is added 2ml Osteoinductive differentiation culture medium and continues Culture, every 3d change 1 not good liquor.After induction differentiation 21d, 4% neutral formalin solution of 2ml, fixed 30min is added in PBS cleaning, every hole. Formalin is discarded, PBS is cleaned 1~2 time.Every hole is added 1ml alizarin red dye liquor and dyes 3~5min, discards dye liquor, PBS cleaning 2 ~3 times.6 orifice plates are placed in microscopically observation dyeing effect and are taken pictures.
Observation result is shown in Fig. 2.The visible calcium deposition of Osteoinductive differentiation cell surface simultaneously forms similar round calcium tubercle, alizarin Red colouring is bronzing, and control group is formed without calcium tubercle, and cultivated cell is prompted to have the Osteoblast Differentiation potential of BMSCs.
Every hole adds 2ml adipogenic induction A liquid, and A liquid is abandoned after 3d and changes 2ml adipogenic induction B liquid, after 1d, abandons B liquid and changes A liquid.A liquid, B Liquid alternating action 3~5 times.4% neutral formalin solution, fixed 30min is added in PBS cleaning.PBS cleaning, oil red O stain 30min, It optical microphotograph sem observation and takes pictures.
The visible round fat drips of Fig. 2 cell cytosol that adipogenic induction breaks up as the result is shown are formed, and oil red O stain is Chinese red, Control group is formed without fat drips, prompts cultivated cell with BMSCs at rouge differentiation potential.
Embodiment 3: the streaming identification of rat bone marrow mesenchymal stem cells.
The P3 cell that Example 1 is cultivated, with its positive surface's marker CD29, CD90 of flow cytomery and feminine gender Surface marker CD45.
The P3 cell for taking degrees of fusion 90% or so siphons away culture medium, and PBS is cleaned 2 times, and it is molten that 0.25% pancreatin without EDTA is added Liquid 3ml, 37 DEG C of digestion 5min, cell rounding floats under microscope, and culture medium is added and terminates digestion.
It is moved into centrifuge tube with suction pipe, 1000rpm is centrifuged 5min.Supernatant is abandoned, PBS is cleaned 2 times, is centrifuged again.It discards Clearly, 1ml PBS is added, cell suspension is made in piping and druming, carries out cell count, cell density 1 × 105A/ml.
Filtration cell, 1000rpm are centrifuged 5min.Discard supernatant, it is 200 μ l that PBS adjustment liquor capacity, which is added, gently with referring to Point bounces cell as suspension, CD29-APC, CD90-FITC, CD45-PE antibody is added, room temperature black out is incubated for 30min.Add Enter 4~5ml PBS washing, 1000rpm is centrifuged 5min, discards supernatant.200 μ l buffers are added, are gently bounced again with finger tip Cell becomes suspension, with flow cytomery P3 for BMSCs surface marker, as a result as shown in Figure 3.
Fig. 3 display BMSCs surface molecular CD29, CD90 expression rate respectively (97.90 ± 0.13) %, (96.00 ± 1.39) %, hematopoietic cell surface marker CD45 expression rate (3.3 ± 0.26) %, result are feminine gender.Cultivated cell positive expression BMSCs specific surfaces molecule, feminine gender expression hematopoietic cell specific surfaces molecule.
Embodiment 4: adenovirus mediatedRspo1Gene-transfected BMSCs.
It takes P3 of the degrees of fusion up to 80~90% for cell, discards culture medium, PBS is cleaned 2 times, and 0.25% pancreatin is added and (contains EDTA) 1.5ml, 37 DEG C of digestion 3min, cells float is rounded under microscope, and culture medium is added and terminates digestion, 1000rpm centrifugation 5min。
Centrifugation finishes, and 1ml culture medium is added and is resuspended, counts.By cell suspension with 8 × 104The density in a/hole is planted in 6 In orifice plate, 37 DEG C, 5% CO2Environment culture is adherent.
It takes out 6 orifice plates afterwards for 24 hours, discards culture medium, adenovirus is added and is transfected.Adenovirus Transfection time 72h, according to disease Malicious titre infection multiplicity (MOI) is 100.
Be divided into 3 groups when adenovirus is added: control group only adds culture medium without double antibody (without penicillin and streptomysin) 2ml;Yin Property control group be added negative control virus liquid (AdV-GFP) 1ml+ culture medium 1ml without double antibody;Concentration 2 × 10 is added in experimental group8 Virus liquid (AdV-GFP-Rspo1) 1ml+ culture medium 1ml without double antibody of PFU/ml.Continue to cultivate 72h.
Fluorescence microscopy microscopic observation transfection results, as Fig. 4 is shown.Control group (Fig. 4 A) has no green fluorescence, negative control Group (Fig. 4 C) and experimental group (Fig. 4 E) visible green fluorescence, it was demonstrated that adenovirus has been transfected into cell.Fig. 4 B, 4D and 4F Fig. 4 A, 4C and the corresponding eucaryotic cell structure figure of 4E is set forth.
Fluorescence microscope cannot tell transfection efficiency, therefore further investigate adenopathy by flow cytomery The transfection efficiency of poison transfection BMSCs.The testing result of Fig. 5 shows that control group transfection efficiency (0.02 ± 0.03) % (Fig. 5 A) is negative Property control group transfection efficiency (99.10 ± 1.25) % (Fig. 5 B), experimental group transfection efficiency (98.50 ± 1.18) % (Fig. 5 C).
Embodiment 5:qPCR detectionRspo1Expression.
Extract the total serum IgE of 3 groups of BMSCs in embodiment 4, DnaseI (DNA respectively with RNA extracts kit Enzyme) RNA sample is handled to remove DNA pollution.
3 groups of RNA samples are diluted to 100 μ g/ml respectively, respectively take 10 μ l samples, synthesize the first chain with reverse transcription reagent box cDNA.Using the first chain cDNA as template, housekeeping geneβ-actinIt is compareed as internal reference, using primer shown in table 1, described in table 2 PCR reaction is carried out according to the reaction condition of table 3 in RT-PCR reaction system.
To PCR reaction band carry out gray scale scanning, respectively withβ-actinGray value compare, according to Real-time PCR Testing result calculates separately out 3 groups of BMSCs'Rspo1Relative expression quantity, as a result as shown in Figure 6.
The results show that experimental groupRspo1Expression quantity be significantly higher than control group (p< 0.01) and negative control group (p< 0.01), show that Rspo1 is expressed by Adenovirus Transfection BMSCs in gene level success height,Rspo1Transfection enters BMSCs.
Embodiment 6:AnnexinV-APC/7-AAD cell box detects rat bone marrow mesenchymal stem cells apoptosis.
The BMSCs of 72h after 3 groups of transfections of Example 4 is digested with the pancreatin without EDTA, collects cell.
It is washed cell 2 times with PBS, 2000rpm is centrifuged 5min.Collect 5 × 105500 μ l Binding are added in cell Buffer suspension cell adds 5 μ l Annnexin V-APC mixing, 5 μ l 7-AAD dye liquors is added, mixes;Room temperature is protected from light 5~15min of lower reaction.Flow cytometer observation and detection are carried out in 1h.
In Fig. 7 testing result, A, B, C are respectively control group, negative control group and experimental group.Statistical result showed, experiment The apoptosis rate of group, negative control group and control group is respectively 25.6%, 42.6% and 37.9%, and experimental group apoptosis rate is bright It is aobvious to be lower than control group and negative control group,Rspo1It is able to suppress the apoptosis of mesenchymal stem cell.
The bis- dyes of embodiment 7:AO-EB detect rat bone marrow mesenchymal stem cells apoptosis.
Using preceding according to dosage, AO solution and EB solution are mixed into working solution by 1: 1, it is ready-to-use.
Adenovirus Transfection mesenchymal stem cell takes control group (no Adenovirus Transfection), feminine gender right respectively with embodiment 4 According to group (transfection AdV-GFP), experimental group (transfection AdV-GFP-Rspo1), remove culture medium.3 groups of cells wash 2 times with PBS Except residual media and non-attached cell, new PBS, every ml is added in control group, negative control group and experimental group respectively 20 μ l working solutions are added in PBS, after being placed at room temperature for 2~5min, under the microscope in fluorescence microscopy.
The nucleus of the mesenchymal stem cell of early apoptosis and late apoptic is rendered as green and Chinese red simultaneously respectively In pyknosis shape or round bead shape.
According to the fluorescence microscope result and statistical result of Fig. 8 can be seen that experimental group apoptosis cell be considerably less than pair According to group and negative control group, showRspo1It can inhibit the apoptosis of mesenchymal stem cell.
The expression of embodiment 8:Rspo1 albumen and apoptosis-related protein Bax, Caspase3 and Cleavage-Caspase3.
Control group, negative control group and experimental group BMSCs cell in embodiment 4 are collected, with total protein extraction kit point The total protein of BMSCs is indescribably taken, BCA method carries out protein quantification.
Every 35 μ g albumen of swimming lane, 10% SDS-PAGE is wet after being separated by electrophoresis to go to pvdf membrane, closes 1h, is added in each group 3 kinds of primary antibody (anti-Bax 1: 1000;anti-Caspase3 1:2000;Anti-Cleavage-Caspase3 1: 1000), and 4 It DEG C is incubated overnight, washes film, add corresponding secondary antibody, be incubated at room temperature 1h, image analysis system gray scale scanning.As a result such as.
Testing result such as Fig. 9.Compared with control group and negative control group, experimental group apoptosis rate is substantially reduced.Experimental group Tri- kinds of protein expression rates of Bax, Caspase3 and Cleavage-Caspase3 be substantially less than control group (Bax,p<0.01; Caspase3,p<0.01;Cleavage-Caspase3,p< 0.01) and negative control group (Bax,p<0.01;Caspase3,p< 0.01;Cleavage-Caspase3,p< 0.01), show that on protein level Rspo1 inhibits the apoptosis of BMSCs.

Claims (7)

1. a kind of utilizationRspo1The method for inhibiting apoptosis of mesenchymal stem cell is to carryRspo1The adenovirus of gene carries Body gene-transfected BMSCs, makeRspo1Into in mesenchymal stem cell, cultivates and obtain in culture medium without double antibody Rotaring redyeing geneRspo1Mesenchymal stem cell.
2. according to the method described in claim 1, it is characterized in that the mesenchymal stem cell is in vitro medulla mesenchyma Stem cell.
3. method according to claim 1 or 2, it is characterized in that the mesenchymal stem cell is between isolated rat marrow Mesenchymal stem cells.
4. according to the method described in claim 3, it is characterized in that using cultivating to the isolated rat mesenchymal stem cell of P3 It is transfected.
5. according to the method described in claim 4, it is characterized in that cell suspension is made in mesenchymal stem cell, 37 DEG C, 5% CO2After cultivating for 24 hours in incubator, Adenovirus Transfection is carried out with MOI=100.
6. according to the method described in claim 4, it is characterized in that by the carryingRspo1The adenovirus vector of gene is configured to dense Degree 2 × 108The adenopathy venom of PFU/ml is with gene-transfected BMSCs.
7. according to the method described in claim 4, it is characterized in that the transfection time is 72h.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007047963A2 (en) * 2005-10-20 2007-04-26 Caritas St. Elizabeth Medical Center Of Boston, Inc. Use of bone-marrow derived stem cells to treat ischemia
CN102329777A (en) * 2011-07-22 2012-01-25 广州医学院第二附属医院 Antiapoptosis high expression hVEGF165 (human Vascular Endothelial Growth Factor 165) cell model and building method thereof
WO2014159356A1 (en) * 2013-03-14 2014-10-02 The Brigham And Women's Hospital, Inc. Compositions and methods for epithelial stem cell expansion and culture
CN106913880A (en) * 2015-12-24 2017-07-04 上海交通大学 A kind of targeting drug delivery system containing RSPO1 and its preparation and application

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007047963A2 (en) * 2005-10-20 2007-04-26 Caritas St. Elizabeth Medical Center Of Boston, Inc. Use of bone-marrow derived stem cells to treat ischemia
CN102329777A (en) * 2011-07-22 2012-01-25 广州医学院第二附属医院 Antiapoptosis high expression hVEGF165 (human Vascular Endothelial Growth Factor 165) cell model and building method thereof
WO2014159356A1 (en) * 2013-03-14 2014-10-02 The Brigham And Women's Hospital, Inc. Compositions and methods for epithelial stem cell expansion and culture
CN106913880A (en) * 2015-12-24 2017-07-04 上海交通大学 A kind of targeting drug delivery system containing RSPO1 and its preparation and application

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHENG,X.X. ET AL: "Apoptosis of mesenchymal stem cells is regulated by Rspo1 via the Wnt/b-catenin signaling pathway", 《CHRONIC DISEASES AND TRANSLATIONAL MEDICINE》 *
DENG,L. ET AL: "Aspirin induces apoptosis in mesenchymal stem cells requiring Wnt/β-catenin pathway", 《CELL PROLIFERATION》 *
WONG,V.S. ET AL: "R-spondin-1 is a novel β-cell Growth Factor and Insulin Secretagogeu", 《JOURNAL OF BIOLOGICAL CHEMISTRY》 *

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Application publication date: 20190405