CN102329777A

CN102329777A - Antiapoptosis high expression hVEGF165 (human Vascular Endothelial Growth Factor 165) cell model and building method thereof

Info

Publication number: CN102329777A
Application number: CN201110206260A
Authority: CN
Inventors: 陈敏生; 林育辉; 倪晓彬; 何晓青; 刘世明; 罗承锋
Original assignee: Second Affiliated Hospital of Guangzhou Medical University
Current assignee: Second Affiliated Hospital of Guangzhou Medical University
Priority date: 2011-07-22
Filing date: 2011-07-22
Publication date: 2012-01-25

Abstract

The invention relates to an antiapoptosis high expression hVEGF165 (human Vascular Endothelial Growth Factor 165) cell model and a building method thereof. The cell model takes BMSCs (Bone Mesenchymal Stem Cells) as transfection cells and coexpresses a recombinant adenovirus with double genes hBCL-2 and hVEGF165. The building method of the cell model comprises the following steps of: 1) separating and purifying rat BMSCs to obtain massive purified BMSCs with good vitality; 2) transfecting the BMSCs by the recombinant adenovirus, building the cell model built by the BMSCs transfected for 72 hours by adopting a multiplicity of infection (MOI) is equal to 400. The application characteristics are as follows: 1) the cell model is used for treating diseases such as myocardial infarction and the like; and 2) the cell model is used for being transplanted into a receptor organ. Compared with common BMSCs, the transfection recombinant adenovirus BMSCs provided by the invention has the advantages that cell apoptosis ratio is obviously reduced, service life is prolonged, the hVEGF165 can be efficiently expressed, the cell model can be conveniently transplanted to the receptor organ, and effect for treating diseases can be improved, thus the cell model and the building method thereof which are provided by the invention have wide application prospect.

Description

A kind of anti-apoptosis efficiently expresses hVEGF 165Cell model and establishment method thereof

Technical field

The present invention relates to a kind of anti-apoptosis and efficiently express hVEGF ₁₆₅Cell model and establishment method thereof.Belong to the biomedical research technical field.

Background technology

The angiogenic action that research shows VEGF partly is that the expression through BCL-2 in the induction of vascular endothelial cell realizes.VEGF induces the high expression level of BCL-2 to help to improve the survival of vascular endothelial cell under conditions such as hypoxemia, poisoning ^[18]The experiment of Xu etc. shows that it mainly is that some realize cardioprotection such as the cytokine of VEGF etc. and then the expression of raising myocardial cell's BCL-2 through paracrine that BMSCs transplants the heart function improve behind the heart stalk ^[19]BCL-2 can also suppress the apoptosis by NO inductive vascular endothelial cell, promotes the formation of blood vessel ^[20]The expression of crossing of BCL-2 has promoted the expression of VEGF and the vasculogenesis of tumour in the tumour cell ^[21]It is thus clear that in various kinds of cell, exist the cascade connection of mutual rise between VEGF and the BCL-2, form synergy on the biological function.BCL-2 and VEGF coexpression combined utilization possibly produce the even more ideal result of treatment of more single therapeutic gene in gene therapy.

The adenovirus carrier host range is wide, transfection efficiency and gene expression dose is high, security is better, is the excellent carrier of gene therapy, is comparatively ideal carrier of myocardial infarction gene therapy.

The gene therapy of myocardial infarction at present or what transplantation treatment myocardial infarction behind the stem cell genetic modification was adopted is monogenic treat-ment such as anti-apoptosis or short vasculogenesis mostly; The report of the dual-gene treatment of rare application, rarer application double gene coexpression carrier.Do not see to choose simultaneously BCL-2 and people VEGF at present as yet ₁₆₅Gene therapy myocardial infarction or to the report of transplantation treatment myocardial infarction behind the stem cell genetic modification.

Mesenchymal stem cells MSCs (BMSCs) content in marrow is low, and its separation and purification problem is that in the BMSCs research is important but also the problem that does not solve fully so far always.At present the method for separation and purification BMSCs has 2 kinds: 1) density gradient centrifugation, BMSCs is different with other cell densities, according to the settlement action principle, is placed in the medium of a certain gradient centrifugal to remove other cells.This density gradient centrifugation can obtain the mesenchymal stem cells MSCs (BMSCs) of relative higher degree, but the active interference of pair cell is bigger.2) surface marker sieve method; Some special surface marks according to cell surface; Carry out the positive or negative screening with various monoclonal antibodies; Because of BMSCs does not have special surface marker, have to adopt multiple affinity tag associating method for screening to carry out, there are selected by flow cytometry apoptosis method, immune lysis method, plane to adhere to partition method, magnetic activated cell seperation at present.Aforesaid method is influential equally to the cytoactive of BMSCs, and need use distinct antibodies or parting liquid, and is more time-consuming and spend more.

The most important step of Adenovirus Transfection cell is that cell surface has acceptor to combine with it, and the adenovirus receptor density and the quantity of transfection efficiency and cell surface are closely related.And adenovirus receptor low expression level or do not express normally on many cell surfaces comprises many tumour cells, Skeletal Muscle Cell, lymphocyte, fibrocyte, hemopoietic stem cell.And behind the Adenovirus Transfection cell, the toxic effect of pair cell can cause the death of cell.Therefore; Want to let recombinant adenovirus can express genes carried and improve the efficient of transfection, at first must select suitable Adenovirus Transfection target cell, and BMSCs often selects as first; But common BMSCs is shorter in the vitro culture life-span, can not continue to efficiently express hVEGF ₁₆₅Therefore, how to make up that a kind of BMSCs of making apoptosis ratio reduces, the life-span prolongs, the time length is longer, efficiently express hVEGF ₁₆₅Cell model be main task of the present invention.

Summary of the invention

First purpose of the present invention is shorter in the vitro culture life-span in order to solve prior art BMSCs, can not continue to efficiently express hVEGF ₁₆₅Problem, provide the good anti-apoptosis of a kind of vigor to efficiently express hVEGF ₁₆₅Cell model.

Second purpose of the present invention is in order to provide a kind of simple and convenient, cost-effective anti-apoptosis to efficiently express hVEGF ₁₆₅The establishment method of cell model.

First purpose of the present invention can reach through following technical scheme:

A kind of anti-apoptosis efficiently expresses hVEGF ₁₆₅Cell model, it is characterized in that: with the rat bone marrow mesenchymal stem cells BMSCs of purifying as transfectional cell, with hBCL-2 and hVEGF ₁₆₅The double gene coexpression recombinant adenovirus is with hBCL-2 and hVEGF ₁₆₅The rat bone marrow mesenchymal stem cells BMSCs of double gene coexpression recombinant adenovirus transfection purifying constitutes anti-apoptosis and efficiently expresses hVEGF ₁₆₅Cell model.

Second purpose of the present invention can reach through following technical scheme:

A kind of anti-apoptosis efficiently expresses hVEGF ₁₆₅The establishment method of cell model, it is characterized in that may further comprise the steps:

1) obtains the rat bone marrow mesenchymal stem cells BMSCs of purifying

Get rat femur; Under aseptic condition, reject muscle tissue, cut off the two ends metaphysis, extract the DMEM substratum that contains 20% foetal calf serum FBS; Wash medullary space repeatedly; The collecting cell suspension carries out centrifugal back supernatant discarded in centrifuge tube, be put in the Tissue Culture Flask behind the adding DMEM substratum, and culturing bottle is placed 35 ℃～39 ℃, 5%CO ₂Cell culture incubator in cultivate; Through rear section cell attachment growth in 20-30 hour, abandoning supernatant was changed liquid 1 time in every then 2-3 days; When cell reaches the 85%-90% fusion; With the cultivation of going down to posterity in 1: 2 or 1: 3, continue to cultivate with the DMEM substratum again, up to obtain purifying rat bone marrow mesenchymal stem cells BMSCs;

2) the rat bone marrow mesenchymal stem cells BMSCs of recombinant adenovirus transfection purifying

The cell suspension density of the rat bone marrow mesenchymal stem cells BMSCs of adjustment purifying is with 5 * 10 ⁴/ ml density is seeded on the porous plate, and every hole adds the DMEM substratum, at cell culture incubator with 35 ℃～39 ℃, 5%CO ₂Condition under cultivate; After 40-50 hour, absorb old nutrient solution, every once more hole adds the DMEM substratum, adds the recombinant adenovirus venom Ad-EGFP-VEGF of respective volume respectively by virus infection plural number MOI=0,10,50,100,200,400 ₁₆₅-BCL-2; Said rat bone marrow mesenchymal stem cells BMSCs cell transfecting, process after 20-30 hour, are discarded old nutrient solution, be changed to the more DMEM nutrient solution of low levels FBS; Continuation is to said rat bone marrow mesenchymal stem cells BMSCs cell transfecting; Different time sections in this transfection process is the expression of observation of cell green fluorescent protein respectively, continues to cultivate after 60-80 hour, digests the porous plate cell then; Every hole adds 0.25% pancreatin 0.5ml, and operation is the same; Be resuspended in behind the cell dissociation with every hole in the flow cytometer dedicated test pipe of the 0.01M PBS that contains 0.5ml; Use suction pipe to blow and beat gently and be the individual cells suspension; On flow cytometer,, record the highest rat bone marrow mesenchymal stem cells BMSCs of transfection efficiency by the ratio of pipe green GFP positive cell;

3) cell model that adopts the highest rat bone marrow mesenchymal stem cells BMSCs of transfection efficiency to set up, anti-apoptosis and efficiently express hVEGF then, ₁₆₅Cell model.

Second purpose of the present invention can also reach through following technical scheme:

Step 1) can be for being described below to rat BMSCs separation and purification process:

Rat is placed in 75% alcohol with 3% vetanarcol 2ml/kg intraperitoneal injection of anesthesia soaks 10min, cut off its fur; Get rat femur, under aseptic condition, reject muscle tissue, cut off the two ends metaphysis; Extract the DMEM substratum that contains 20% foetal calf serum FBS with the 10ml syringe, wash medullary space repeatedly, the collecting cell suspension is in centrifuge tube; With the centrifugal 10min of 1200rpm/min, supernatant discarded adds the DMEM substratum that contains 10%FBS; Blow and be put in the Tissue Culture Flask after even, culturing bottle is placed 35 ℃～39 ℃, 5%CO ₂Cell culture incubator in cultivate; Through behind the 24h, part cell adherent growth, abandoning supernatant was changed liquid 1 time in every then 2-3 days, when cell reaches the 85%-90% fusion,, cultivates with the DMEM substratum continuation of 10%FBS with the cultivation of going down to posterity in 1: 2 or 1: 3, thus the BMSCs of acquisition large-scale purification;

Step 2) recombinant adenovirus transfection BMSCs process can be described below:

With the density of cell counter adjustment BMSCs cell suspension, with 5 * 10 ⁴/ ml density is seeded on 6 orifice plates, and every hole adds the DMEM substratum that 1.5ml contains 10%FBS, at cell culture incubator with 35 ℃～39 ℃, 5%CO ₂Condition under cultivate; Through behind the 48h, absorb old nutrient solution, every hole adds the DMEM of 1.5ml, adds the recombinant adenovirus venom Ad-EGFP-VEGF of respective volume respectively by virus infection plural number MOI=0,10,50,100,200,400 ₁₆₅(titre is 5 * 10 to-BCL-2 ⁹Pfu/ml); Behind the transfection 24h, discard old nutrient solution, be changed to the DMEM nutrient solution that contains 2%FBS,, express the strongest when the result draws 72h through 24h, 48h, the 72h expression of observation of cell green fluorescent protein under fluorescent microscope respectively; Continue to cultivate 72h, digest 6 orifice plate cells then, every hole adds 0.25% pancreatin 0.5ml, and operation is the same; Be resuspended in behind the cell dissociation with every hole in the flow cytometer dedicated test pipe of the 0.01M PBS that contains 0.5ml; Use suction pipe to blow and beat gently and be the individual cells suspension; On flow cytometer by the ratio of pipe green GFP positive cell, thereby transfection efficiency is the highest when recording MOI=400; Then, adopt the BMSCs of MOI=400 transfection 72h to set up cell model.

Rat can be the SD rat described in the step 1) step, heavy 100-150g.

Tissue Culture Flask can be 2 T-25cm described in the step 1) step ²Tissue Culture Flask.

Step 2) expression of observation of cell green fluorescent protein described in the step can be passed through common light microscopic or fluorescence microscope.

Anti-apoptosis according to the invention and efficiently express hVEGF ₁₆₅The purposes of cell model, 1) is used to treat diseases such as myocardial infarction; 2) be used to migrate to the acceptor organ.

The present invention has following outstanding beneficial effect:

1, the present invention filters out BMSCs as hBCL-2 and hVEGF ₁₆₅Double gene coexpression recombinant adenovirus ideal transfection target cell, determine the transfection recombinant adenovirus after BMSCs efficiently secrete hVEGF ₁₆₅And deprive the reduction of the BMSCs apoptosis ratio after the transfection under the environment at simulation serum; HBCL-2 and hVEGF have been solved ₁₆₅The problem that the double gene coexpression recombinant adenovirus is difficult for transfectional cell and expresses in target cell the inside, after also having solved common BMSCs and being transplanted to the hypoxic-ischemic heart life-span shorter, can not continue to efficiently express hVEGF ₁₆₅Problem; This cell model will be opened up new road in cellular replacement therapy myocardial infarction field, have potential applicability in clinical practice widely.

2, the present invention through the separation and purification of improvement can be fast good BMSCs of acquisition vigor in a large number, transfection efficiency reaches 70%, for diseases such as BMSCs cellular transplantation therapy myocardial infarction clinically facilitate.

3, the apoptosis ratio of the more common BMSCs of transfection recombinant adenovirus BMSCs of the present invention has obvious minimizing, and the life-span prolongs, and can efficiently express hVEGF ₁₆₅, help this cell model is migrated to the acceptor organ, and can improve the effect of treatment disease, be with a wide range of applications.

Description of drawings

Figure 1A-1C is the former rat BMSCs figure that is commissioned to train foster.

Fig. 1 D-1F is the rat BMSCs figure that goes down to posterity and cultivate.

Fig. 2 A-2H is a BMSCs cell surface marker logistics formula cell instrument detected result synoptic diagram.

Fig. 3 A, 3C, 3E are common light microscopic 24h, 48h, the 72h variation diagram of the BMSCs behind the transfection recombinant adenovirus.

Fig. 3 B, 3D, 3F are the fluorescent microscope variation diagram of the BMSCs behind the transfection recombinant adenovirus.

Fig. 4 A-4F is a cells were tested by flow cytometry recombinant adenovirus transfection BMSCs efficient synoptic diagram.

Fig. 5 is VEGF ₁₆₅The secretion level synoptic diagram.

Fig. 6 A-6C detects the diffusing point of a flow cytometer synoptic diagram for the BMSCs apoptosis.

Embodiment

Specific embodiment 1:

The anti-apoptosis that present embodiment relates to and efficiently express hVEGF ₁₆₅Cell model, with the rat bone marrow mesenchymal stem cells BMSCs of purifying as transfectional cell, with hBCL-2 and hVEGF ₁₆₅The double gene coexpression recombinant adenovirus is with hBCL-2 and hVEGF ₁₆₅The rat bone marrow mesenchymal stem cells BMSCs of double gene coexpression recombinant adenovirus transfection purifying constitutes anti-apoptosis and efficiently expresses hVEGF ₁₆₅Cell model.

The establishment method and the testing process of present embodiment may further comprise the steps: the rat bone marrow mesenchymal stem cells BMSCs that 1) obtains purifying; 2) the rat bone marrow mesenchymal stem cells BMSCs of recombinant adenovirus transfection purifying; 3) cell model that adopts the highest rat bone marrow mesenchymal stem cells BMSCs of transfection efficiency to set up, anti-apoptosis and efficiently express hVEGF then, ₁₆₅Cell model.

Concrete operation method is following:

One, to rat BMSCs separation and purification

1, the separation of rat BMSCs

1) get the male SD rat of 100-150g, 3% vetanarcol 2ml/Kg intraperitoneal injection of anesthesia is placed in 75% alcohol and soaked 10 minutes, then mouse is placed in the plastic tub of sterilizing.

2) wear sterile gloves, cut off SD rat skin of lower extremity, expose lower limb muscles fully, passivity separating muscle tissue appears femur and shin bone, carefully separates complete taking-up femur with the shin bone junction from femur; Take out shin bone with method, be transferred to Bechtop then, be put in the petridish that contains aseptic 0.01M foetal calf serum FBS.

3) isolating SD rat femur and shin bone are placed the aseptic foetal calf serum FBS of 10ml petridish is housed, the sterile gloves that renews is used the aseptic nipper and the scissors of super clean bench the inside instead, rejects fat and muscle tissue, exposes the marrow end.Be put into another one then and contain the blood of removing the bone outside in the petridish of aseptic foetal calf serum FBS and the muscle that adheres to, wash repeatedly three times with aseptic foetal calf serum FBS, no blood and muscle are residual outside bone.

4) cut off the cartilage at bone two ends with clipper; Mosquito forceps is clamped bone; Extract the H-DMEM substratum 5ml that contains 20% foetal calf serum FBS with the 10ml syringe, syringe needle is inserted flushing repeatedly in the medullary space, visible bone marrow fluid is cloud from the cartilage end and flows out; Treat to stop when medullary space bleaches flushing, obtain the marrow of all the other bones with method.

5) collect the marrow suspension in the 15ml centrifuge tube, the centrifugal 10min of 1200rpm room temperature abandons supernatant, contains 20% foetal calf serum FBS fresh culture re-suspended cell with 8ml, is transferred to 2 T-25cm ²In the Tissue Culture Flask.

2, rat BMSCs former is commissioned to train foster

Tissue Culture Flask is placed 37 ℃, 5%CO ₂Cultivate in the cell culture incubator of saturated humidity, the mononuclearcell that volume is bigger in the marrow when rigidly connecting kind is spherical in shape, is suspended in the nutrient solution, and it is adherent that 8-10h begins sedimentation gradually; Discard not attached cell behind the 24h, change nutrient solution, with the H-DMEM culture medium culturing cell that contains 10% foetal calf serum FBS, visible cell is adherent basically, but cell quantity is less, and the elongated or polygon of cell is shown in Figure 1A; Changed liquid 1 time in later every 2-3 days, and be cultured to the 3rd day visible cell and how become spindle shape gradually, shown in Figure 1B; Cell fast breeding afterwards, nucleus is placed in the middle, and idol has double-core, and the 6th day cell forms colony to the inoculation back, and likeness in form corynebacterium or fiber-like are parallel or swirl shape is arranged, shown in Fig. 1 C; After attached cell is merged near 80%-90%, finish former be commissioned to train foster.

3. the rat BMSCs amplification cultivation that goes down to posterity

When 1) cytogamy is to 80%-90%, discard old nutrient solution, add the foetal calf serum FBS solution of 2ml sterilization 0.01M, rock gently, the washed cell aufwuchsplate discards PBS solution then.

2) add 1ml 0.25% pancreatin, place observation of cell form under the inverted microscope, after treating under the mirror that projection disappears between the cell, becoming circle, discard pancreatin; Add the H-DMEM cell culture medium 6ml that contains 10% foetal calf serum FBS and stop digestion, softly blow and beat into single cell suspension with suction pipe.

3) by 1: 2 branch to 2 new T-25cm that goes down to posterity ²Culturing bottle places 37 ℃, 5%CO ₂Incubator in continue to cultivate, be designated as P1 (the 1st generation) this moment, later every 3-4 days passage once.The cultivation 3-4h that goes down to posterity begins adherent, and 6-8h is adherent fully, prepares against experiment time spent cellular form to P1, P2, P3 and does not see considerable change; But it is more rapid to grow; At the bottom of covering with sooner in the same time bottle, uniform sequentially with inoblast appearance distribution characteristics, shown in Fig. 1 D-1F.

4. the evaluation of rat BMSCs

1) about P3 cytogamy to 90%, use 0.25% trypsin digestion cell, concrete steps are the same.

2) collecting cell suspension, behind the cell counter counting, adjusting each cell count that detects sample is 5 * 10 ⁵

3) the 1200rpm room temperature is centrifugal 5 minutes, abandons supernatant.

4) sterilization foetal calf serum FBS washing is 2 times and centrifugal under the same terms; Abandon supernatant; In sample to be detected, respectively add 100ul sterilization 1 * foetal calf serum FBS solution, mixing adds CD29, CD34, CD44, CD45, CD90 one anti-and each 10ul of homotype negative control respectively gently.

5) 37 ℃ of lucifuges were hatched 15 minutes, and the upflowing cell instrument detected after every pipe added foetal calf serum FBS solution 1ml.Detected result is shown in Fig. 2 A-2H.

Two, recombinant adenovirus transfection BMSCs

1, the mensuration of recombinant adenovirus transfection BMSCs efficient

1) digests the density of cell suspension afterwards with the cell counter adjustment, with 5 * 10 ⁴/ ml density is seeded in 6 orifice plates, every hole add 1.5ml contain 10% foetal calf serum FBS DMEM at cell culture incubator with 5%CO ₂, cultivate under 37 ℃ of conditions.

2) absorb old nutrient solution behind the cultivation 24h, every hole still adds the DMEM substratum that 1.5ml contains 10% foetal calf serum FBS, continues in incubator, to cultivate.

3) behind the 24h, absorb old nutrient solution, every hole adds 1.5ml DMEM substratum.(multiplicity ofinfection MOI), adds the reorganization Ad-EGFP-VEGF of respective volume respectively by MOI=0,10,50,100,200,400 in order to obtain best virus infection plural number ₁₆₅(titre is 5 * 10 to-BCL-2 ⁹Pfu/mL) viral liquid.

4) discard old nutrient solution behind the transfection 24h, be changed to the DMEM substratum that contains 2% foetal calf serum FBS,, reach the strongest when the result is illustrated in 72h, no longer increase thereafter, shown in Fig. 3 A-3F through 24h, 48h, the 72h expression of observation of cell green fluorescent protein respectively.

5) continue to cultivate 72h, digest 6 orifice plate cells then, every hole adds 0.25% pancreatin 0.5ml, and operation is the same; Be resuspended in behind the cell dissociation with each hole in the flow cytometer dedicated test pipe that contains 0.5ml 0.01M PBS, use suction pipe to blow and beat gently and be the individual cells suspension.

6) on flow cytometer, examine the ratio of green fluorescent protein positive cell, record its transfection efficiency between 8.08%～70.65%, can know best MOI=400, shown in Fig. 4 A-4F by managing detection.

Three, ELISA detects after the transfection VEGF in the BMSCs culture supernatant ₁₆₅Content

1, the collection of BMSCs culture supernatant liquid

1) peptic cell is inoculated in 6 orifice plates, and with viral liquid transfection BMSCs.

2) be divided into 3 groups: the 1st group is normal BMSCs group; The 2nd group is the BMSCs group of transfection Ad-EGFP; The 3rd group is transfection Ad-EGFP-VEGF ¹⁶⁵The BMSCs group of-BCL-2.After transfection virus, collected the cell conditioned medium liquid of culture hole on the 1st, 3,5,7,9,11,13,15,17 day, collected supernatant at every turn after, every hole adds the fresh DMEM substratum of 1.5ml more again.Until the 15th day.Supernatant after the collection is with 3000rpm/min, and 4 ℃ of centrifugal 20min are put in-20 ℃ of refrigerators then and preserve.After waiting to have collected the cell conditioned medium liquid of all time points, each sample is detected simultaneously.

2.ELISA test kit detects VEGF in the supernatant ₁₆₅Concentration

1) dilution of standard substance and application of sample: encapsulate at enzyme mark and to establish 10 holes, standard substance hole on the plate, in the 1st, the 2nd hole, add standard substance 100ul respectively, in the 1st, the 2nd hole, add standard substance diluent 50ul, mixing then; From the 1st, the 2nd hole, respectively get 100ul then and be added to the 3rd hole and the 4th hole respectively, add standard substance diluent 50ul, mixing respectively in the 3rd, the 4th hole again; In the 3rd hole and the 4th hole, respectively get 50ul earlier then and discard, respectively get 50ul again and join respectively in the 5th, the 6th hole, in the 5th, the 6th hole, add standard substance diluent 50ul, mixing more respectively; From the 5th, the 6th hole, respectively getting 50ul behind the mixing is added to respectively in the 7th, the 8th hole; In the 7th, the 8th hole, add standard substance diluent 50ul more respectively; From the 7th, the 8th hole, getting 50ul behind the mixing respectively is added in the 9th, the 10th hole; Add standard substance diluent 50ul respectively in the 9 10th hole again, respectively get 50ul from the 9 10th hole behind the mixing and discard.(each hole application of sample amount of dilution back is 50ul all, and concentration is respectively 600pg/ml, 400pg/ml, and 200pg/ml, 100pg/ml, 50pg/ml).

2) application of sample: establish blank well (the blank hole does not add sample and enzyme marking reagent, and all the other each step operations are identical), testing sample hole respectively.Encapsulate at enzyme mark and to add sample diluent 40ul on the plate in the testing sample hole earlier, and then add testing sample 10ul (the final extent of dilution of sample is 5 times).Application of sample is added on bottom, enzyme plate hole with sample, does not touch hole wall as far as possible, rocks mixing gently.

3) incubation: be placed on 37 ℃ of incubations 30 minutes with shrouding film shrouding.

4) dosing: 30 times of concentrated cleaning solutions are subsequent use after with 30 times of dilutions of zero(ppm) water.

5) washing: carefully take the shrouding film off, discard liquid, dry, washings is filled it up with in every hole, leaves standstill after 30 seconds to discard, and so repeats 5 times, claps and does.

6) enzyme-added: every hole adds enzyme marking reagent 50ul, except the blank well.

7) incubation: operation is with 3) step.

8) washing: operation is with 5) step.

9) colour developing: every hole adds developer A50ul earlier, adds developer B50ul again, the mixing that vibrates gently, and 37 ℃ of lucifuges developed the color 15 minutes.

10) stop: every hole adds stop buffer 50ul, termination reaction.

11) measure: with the blank well zeroing, the 450nm wavelength is measured the absorbancy (OD value) in each hole in regular turn.Mensuration should be after adding stop buffer be carried out with interior in 15 minutes.

12) drawing standard curve, SPSS10.0 software match quadratic equation converts sample OD value into concentration.

The VEGF of BMSCs behind the transfection recombinant adenovirus ₁₆₅(pg/ml) is as shown in Figure 5 for secretion level, except 1d, 3d, 7d and 11d, and transfection Ad-EGFP-VEGF ₁₆₅The secretion level of VEGF165 is compared with normal BMSCs group in the BMSCs supernatant of-BCL-2 adenovirus, is significantly increased, and difference has statistical significance (P＜0.05); Except 1d and 3d, all the other time points are compared VEGF with the BMSCs group of transfection Ad-EGFP ₁₆₅Secretion level significant difference (P＜0.05) is arranged; Normal group and transfection Ad-EGFP group VEGF ₁₆₅Secretion level difference not statistically significant (P＞0.05).As time goes on, transfection Ad-EGFP-VEGF ₁₆₅-BCL-2 adenovirus group VEGF ₁₆₅Secretory volume increases gradually, to 13d (924.3 ± 56.5) pg/ml that peaks, n=3.After this, VEGF ₁₆₅Secretion descends gradually, but but still remain on the detection level, and continue to after 17d (4682 ± 83.3) pg/ml.BMSCs group (494.6 ± 114.8) pg/ml of transfection Ad-EGFP and normal BMSCs group (592.0 ± 135.8) pg/ml reach the secretion peak value in 11d.

Four, BMSCs deprives apoptosis detection under the environment at simulation serum behind the transfection recombinant adenovirus

1, detecting box pair cell apoptosis with Annexin V-PE/7-AAD detects

2) be divided into 3 groups: the 1st group is normal BMSCs group; The 2nd group is the BMSCs group of transfection Ad-EGFP; The 3rd group is the BMSCs group of transfection Ad-EGFP-VEGF165-BCL-2.

3) behind transfection virus 24h, the cell of each group to remove residual substratum, adds the H-DMEM substratum that does not contain FBS with 0.01MPBS rinsing 2 times, cultivates 6 days down with the serum-free environment.

4) absorb the old nutrient solution of culture hole the inside, each hole is with the foetal calf serum FBS rinsing of 0.01M one time.

5) every hole adds 0.25% trypsin digestion cell 1min, and the application of sample rifle is absorbed pancreatin gently.

6) every hole adds 1ml foetal calf serum FBS solution, with the attached cell of the inside, suction pipe piping and druming hole, then single cell suspension is transferred to the 15ml centrifuge tube.

7) 1200rpm/min, 4 ℃ of centrifugal 5min.

8) add 100ul 1 * Binding Buffer, blow and beat into single cell suspension gently, transfer to flow cytometer dedicated test pipe then with suction pipe.

9) each detector tube adds 5ul Annexin V-PE and 5ul 7-AAD successively.

10) oscillation test pipe gently, lucifuge reaction 15min under the room temperature.

11) each pipe adds 400ul 1 * Binding Buffer again, and mixing gently vibrates.

12) in 1h on flow cytometer machine testing.

Shown in Fig. 6 A-6C, be respectively the 1st group, the 2nd group and the 3rd group of BMSCs apoptosis detection flow cytometer scatter diagram, left lower quadrant shows viable cell, is (PE-/AAD-); Right upper quadrant is non-viable cell, i.e. necrosis and non-viable apoptotic cell are (PE+/AAD+); And right lower quadrant is a viable apoptotic cell, manifests (PE+/AAD-), can see that the 3rd group of cell left lower quadrant cell count is few, and the apoptotic cell ratio is low, and the 1st group and the 2nd group of cell left lower quadrant apoptotic cell ratio are higher.

The apoptosis rate (3.43 ± 1.61%) of the 3rd group of cell is obviously low than the 2nd group (9.92 ± 5.99%) and the 1st group (10.51 ± 4.54%), and significant difference (p＜0.05) is arranged.The 1st group of apoptosis rate difference with the 2nd group does not then have statistical significance (p＞0.05).BMSCs expressed the increase of BCL-2 albumen after the result that apoptosis detects proved the transfection recombinant adenovirus, can suppress the apoptosis of cell, reduced the apoptotic cell ratio, had actual biological effect.As shown in table 1 below.

The apoptosis rate of the different group BMSCs of table 1

(annotate: compare with the 2nd group, ^*P＜0.05; Compare #p＜0.05 with the 1st group; Compare △ p＞0.05 with the 1st group for the 2nd group.)

DMEM substratum noted earlier, its English full name are exactly Dulbecco ' s Modified Eagle Media, and Chinese full name is exactly a Dulbecco improvement Eagls nutrient solution.

Digestion noted earlier is meant after being grown in the in blocks or agglomerating BMSCs cell in cell plate surfaces and adding 0.25% trypsin solution effect, becomes single cell suspension.This is that the single cell suspension that often needs individual cells to mix in the time of will doing subsequent experimental uses trysinization to be individual cells usually because of cell agglomerating growth of easy multilayer when vitro culture.Concrete steps are exactly that the old nutrient solution (DMEM that contains 10% foetal calf serum) of culture hole the inside is absorbed with liquid-transfering gun, with culture hole of PBS liquid flushing, absorb the PBS after washing again then; Add 0.25% pancreatin then; Digest after 1 minute, absorb pancreatin, add fresh nutrient solution or PBS at last; Cell with at the bottom of the suction pipe piping and druming culture hole just can obtain single cell suspension.

Foregoing BMSCs is meant rat bone marrow mesenchymal stem cells BMSCs.

Claims

1. an anti-apoptosis efficiently expresses hVEGF ₁₆₅Cell model, it is characterized in that: with the rat bone marrow mesenchymal stem cells BMSCs of purifying as transfectional cell, with hBCL-2 and hVEGF ₁₆₅The double gene coexpression recombinant adenovirus is with hBCL-2 and hVEGF ₁₆₅The rat bone marrow mesenchymal stem cells BMSCs of double gene coexpression recombinant adenovirus transfection purifying constitutes anti-apoptosis and efficiently expresses hVEGF ₁₆₅Cell model.

2. a kind of anti-apoptosis as claimed in claim 1 efficiently expresses hVEGF ₁₆₅The establishment method of cell model, it is characterized in that may further comprise the steps:

1) obtains the rat bone marrow mesenchymal stem cells BMSCs of purifying

3. a kind of anti-apoptosis as claimed in claim 2 efficiently expresses hVEGF ₁₆₅The establishment method of cell model, it is characterized in that: in the step 1), before will getting rat femur, rat is placed in 75% alcohol with 3% vetanarcol 2ml/kg intraperitoneal injection of anesthesia soaks 10min earlier, cut off its fur; The DMEM substratum of used flushing medullary space is the DMEM substratum that contains 20% foetal calf serum FBS; Be used for culturing cell the DMEM substratum be the DMEM substratum that contains 10% foetal calf serum FBS.

4. a kind of anti-apoptosis as claimed in claim 2 efficiently expresses hVEGF ₁₆₅The establishment method of cell model; It is characterized in that: in the step 1); The DMEM substratum that is used for the culturing cell first time is the DMEM substratum that contains 10% foetal calf serum FBS, and being used for for the second time, the DMEM substratum of culturing cell is the DMEM substratum that contains 2% foetal calf serum FBS.

5. a kind of anti-apoptosis according to claim 2 and efficiently express hVEGF ₁₆₅The establishment method of cell model is characterized in that: 1) rat described in the step is the SD rat, heavy 100-150g.

6. a kind of anti-apoptosis according to claim 2 efficiently expresses hVEGF ₁₆₅The establishment method of cell model is characterized in that: 1) Tissue Culture Flask described in the step is two T-25cm ²Tissue Culture Flask.

7. a kind of anti-apoptosis according to claim 2 efficiently expresses hVEGF ₁₆₅The establishment method of cell model is characterized in that: 2) expression of the observation of cell green fluorescent protein described in the step is through common light microscopic or fluorescence microscope.