CN107557389B - Method for constructing microencapsulated hBMP-2/Tet-on system - Google Patents

Abstract

The invention provides a method for constructing a microencapsulated hBMP-2/Tet-on system, which comprises the following steps: s1, obtaining hBMP-2 gene, and constructing hBMP-2-GV347 recombinant plasmid; s2, transforming and amplifying the hBMP-2-GV347 recombinant plasmid, infecting cells, inducing, and packaging by using a lentiviral vector to obtain the BMSCs transfected by the gene; s3, encapsulating the BMSCs transfected by the gene obtained in the S2 by using microcapsules; the invention constructs the Tet-On system and the hBMP-2 On the lentiviral vector simultaneously, can improve the transfection efficiency and expression of the two systems, and can express the target gene more stably and continuously. After BMSCs are transfected, stable expression of BMP-2 protein can be obtained under the action of doxycycline, and BMSCs are promoted to be differentiated into osteoblasts. A carboxymethyl cellulose microcapsule containing a phenol group is adopted for wrapping to form a complex, so that the system can stably and continuously secrete BMP-2 protein to the outside of the capsule under the action of DOX; BMP-2 secretion stops after DOX is stopped. BMSCs cells in the microcapsule still have osteogenic differentiation capacity after the capsule is broken, and can be subjected to osteogenic differentiation under the action of autocrine BMP-2.

Description

Method for constructing microencapsulated hBMP-2/Tet-on system

Technical Field

The invention belongs to the technical field of bone tissue engineering, and relates to a method for constructing a microencapsulated hBMP-2/Tet-on system.

Background

Bone grafting is the gold standard for treating bone defects and bone nonunion, and the bone grafting materials commonly used in clinic at present comprise autologous bones, allogeneic bone grafting and tissue engineering bones. The autologous bone source is less, the allogeneic bone cost is expensive, and the risks of disease transmission and immunological rejection exist, so that the method has limitations. The development of tissue engineering technology provides a new direction for the repair of large bone defects. The tissue engineering bone scaffold material has wide sources, and can promote bone regeneration by combining with seed cells.

Bone Morphogenetic Protein (BMP), a specific substance that forms bone tissue, can induce the directional differentiation of undifferentiated mesenchymal cells (BMSCs) into osteocytes. Among them, BMP-2 is the most widely used osteogenesis inducing factor, and it induces ectopic osteogenesis efficiently and stably. Researches show that the bone repair material added with rhBMP-2 or bone mesenchymal stem cells over expressing the BMP-2 can improve the repair speed of bone defect and increase the number of trabeculae and the cortical bone strength in an animal bone defect model. This suggests that local expression of large amounts of BMP promotes osteogenesis and that short-term in vitro elevation of BMP-2 levels is beneficial for therapy. However, the amount of BMPs secreted by human body is very limited, the biological half-life period of the BMPs is short, and exogenous injection of BMP-2 cannot maintain the effective concentration of the BMPs. Therefore, the BMP-2 gene is introduced by using the gene recombination technology, so that an organism can generate continuous and stable endogenous BMP-2 protein, and the bone tissue repair can be promoted. Long-term BMP-2 secretion carries a certain biological risk, and becomes a difficult point for controlling the expression of the BMP-2 gene while introducing the gene. The Qiandongyang and the like respectively construct adenovirus vectors containing an hBMP-2 target gene and a Tet-On system, and the hBMP-2 shows significant expression under the induction of low-dose DOX after the system co-infects BMsCs. Alternative genetic vectors include viral vectors and non-viral vectors, which are incapable of integrating the hBMP-2 genome into the genome of eukaryotic cells. Adenovirus is most widely used, but after adenovirus is modified, the immune response and cytotoxicity of virus gene expression products still remain, and adverse factors are brought to local bone defect treatment. Lentivirus (lentivirus) vectors are gene vectors developed based on HIV-1 (human immunodeficiency virus I), and are developed from the first generation to the fourth generation, most of pathogenic genes are deleted, only gag, pol and rev genes are reserved, most of the viruses are in transcriptional inactivation, and even if genome recombination occurs, the viruses cannot be further replicated and diffused, so that the safety of the lentiviruses as the vectors is greatly improved.

In the process of constructing tissue engineering bone, it is very important to obtain seed cells expressing target gene vectors. Bone marrow mesenchymal stem cells (BMSCs) are pluripotent stem cells existing in bone marrow, have the potential to differentiate in multiple directions, such as chondroblasts and osteoblasts, and are easily transfected and expressed by foreign genes. Based on these characteristics, BMSCs are a good seed cell. However, cells which are not self-derived are easy to cause immunological rejection, and the use of microcapsules to encapsulate cells is a feasible method for isolating immune response. Lim and the like wrap islet cells in a sodium alginate-polylysine-polyethyleneimine membrane, and the islet cells are transplanted into the abdominal cavity of a diabetes model rat in the same way to maintain normal blood sugar for 3 weeks, and the research marks the establishment of a microencapsulation cell transplantation technology. The Li Bao high-voltage electrostatic field method is utilized to prepare APA microcapsules by using Dinghui, etc., BMSCs containing BMP-2 gene are encapsulated in the microcapsules, until 30 days after the microcapsules are encapsulated, BMP-2 protein can still be detected in the supernatant of the culture solution, and after BMP-2 gene-transferred microencapsulated cells and extracapsular bone marrow mesenchymal stem cells are co-cultured for 10 days, the ALP activity quantitative detection result shows that ALP activity of hBMP-2 transfected cell group is increased. The microcapsule material must have good mechanical stability and biocompatibility and a low source of immunity; the proper permeability is beneficial to the release of bioactive factors generated by the cells in the capsule to the outside of the capsule, and can ensure the full absorption of oxygen and nutrient substances and the excretion of metabolites thereof by the cells, and meanwhile, macromolecular immune molecules such as complement and the like can not enter the microcapsule; the microcapsule has uniform size and moderate diameter, and the transplanted cells are completely encapsulated in the microcapsule and do not protrude out of the surface of the microcapsule. The carboxymethyl cellulose (CMC-Ph) containing phenol group is ionic water-soluble cellulose ether, is harmless to human body, has good biodegradability and biocompatibility, and is a good microcapsule material. Carboxyl and hydroxyl on a CMC molecular chain provide a large number of chemically modified sites, a microfluid device is designed by Koshiyu and the like, phenol groups are condensed by using horseradish peroxidase catalysis, the cross-linking and curing of CMC-Ph are realized, and a monodisperse CMC-Ph microcapsule is prepared, can achieve uniformity and stability after being wrapped by fibroblasts, and has unchanged cell proliferation activity.

Disclosure of Invention

The invention aims to provide a method for constructing a microencapsulated hBMP-2/Tet-on system according to the defects in the prior art.

The study uses lentivirus as a vector of the hBMP-2 gene, and has the advantages that: the cell can be infected with the insertable segment in any period, has high expression safety, small inactivation toxicity, easy operation and integration into a target genome, and the obtained stable cell strain has higher infection efficiency than adenovirus.

The technical purpose is realized by the following technical scheme:

a method for constructing a microencapsulated hBMP-2/Tet-on system comprises the following steps:

s1, designing a target gene hBMP-2 primer, directionally cloning the target gene hBMP-2 to a GV347 vector carrying a Tet-on regulation system after PCR amplification and purification, and constructing a hBMP-2-GV347 plasmid;

s2, transforming and amplifying the hBMP-2-GV347 plasmid, and co-infecting 293T cells with the helper plasmid; collecting and concentrating lentivirus liquid, and transfecting 293T cells by using a lentivirus vector to obtain a lentivirus vector containing hBMP-2-GV 347;

s3, transfecting BMSCs with the lentivirus vector containing hBMP-2-GV347 to obtain hBMP-2-GV 347-BMSCs;

s4, encapsulating hBMP-2-GV347-BMSCs by using carboxymethyl cellulose containing phenolic groups as a microcapsule material.

Wherein the particle size of the phenolic carboxymethyl cellulose microcapsule in S4 is 80-160 μm.

Wherein, the coaxial copolymerization microfluid is used for preparing the phenol carboxymethyl cellulose microcapsule, and the preparation method of the phenol carboxymethyl cellulose microcapsule comprises the following steps: the method comprises the steps of using a liquid copolymerization microfluid device and a micro-injection pump as generators, using liquid paraffin as external fluid, using a carboxymethyl cellulose solution containing phenol groups as internal fluid, standing a phenol-based carboxymethyl cellulose microcapsule for 20-30min, discarding the liquid paraffin after the microcapsule is completely deposited at the bottom of a collecting pipe, adding a PBS solution, and performing centrifugal washing on a centrifugal machine at 2000rpm/min multiplied by 5 min.

Wherein the microcapsule package in S4 specifically comprises: selecting hBMP-2-GV347-BMSCs 1 x 10⁷The cell suspension is digested for standby; dispersing phase is 0.1g of phenolic carboxymethyl cellulose, dissolving in 4ml of L-DMEM, and magnetically stirring for 30min until completely dissolving; adding 1mg/mL horseradish peroxidase, and fully dissolving; adjusting the flow rate on the micro pump: the dosage of the continuous phase is 16 mL/min; the dosage of the dispersed phase is 50 mu L/min; repeatedly centrifuging, standing and washing.

Compared with the prior art, the invention has the following advantages and beneficial effects:

the Tet-On system and the hBMP-2 are simultaneously constructed On the lentiviral vector, and compared with the prior method of respectively constructing two adenoviral vectors, the transfection efficiency and expression of the two systems can be improved, and the target gene can be more stably and continuously expressed. After BMSCs are transfected, stable expression of BMP-2 protein can be obtained under the action of doxycycline, and BMSCs are promoted to be differentiated into osteoblasts. By utilizing the micro-fluid theory, the carboxymethyl cellulose microcapsule containing the phenol group is successfully constructed, so that the carboxymethyl cellulose microcapsule has a uniform diameter (80-160 mu m) and has mechanical strength superior to that of a sodium alginate microcapsule. The survival rate of the hBMP-2-GV347-BMSCs in the CMC-Ph microcapsule complex is higher than that of the APA microcapsule complex, and the microcapsule cell complex can stably and continuously secrete BMP-2 protein out of the capsule under the action of DOX (doxycycline); BMP-2 secretion stops after DOX is stopped. BMSCs cells in the microcapsule still have osteogenic differentiation capacity after the capsule is broken, and can be subjected to osteogenic differentiation under the action of autocrine BMP-2.

Drawings

FIG. 1 shows the construction of lentivirus containing recombinant plasmid and the titer detection process.

FIG. 2 agarose gel electrophoresis image after primer PCR.

FIG. 3 shows the result of the cleavage of the GV347 vector.

FIG. 4 Western blot detection of BMP-2 expression in 293T cells.

FIG. 5 is a graph showing the results of electrophoresis.

FIG. 6 different MOI values of lentiviruses infect BMSCs (A: 10; B: 30; C:50) (. times.100).

FIG. 7 shows the proliferative capacity of cells after transfection of 1, 4 and 7d for WST-8 proliferation assay.

FIG. 8 quantitative PCR detection of BMP2 gene expression in different BMSCs.

FIG. 9 Western-blot was used to detect the expression of BMP-2 protein in different BMSCs.

FIG. 10 quantitative PCR detection of RUNX2 gene expression in different BMSCs.

FIG. 11 quantitative PCR detection of OCN gene expression in different BMSCs.

Fig. 12 is a graph showing the relationship between the microcapsule size and the flow rate of liquid paraffin.

FIG. 13 is an under-the-mirror image of microencapsulated cells.

FIG. 14 is a dead and live staining of cells within microcapsules after 24 hours of culture.

FIG. 15 shows the proliferation of cells in the microcapsules after 2 weeks of culture.

FIG. 16 shows the proliferation of cells within the microcapsules.

FIG. 17 is a comparison of BMP2/Tet-on-BMSCs cell secretion in microcapsules and BMP-2 protein secretion from BMP2/Tet-on-BMSCs without microencapsulation.

FIG. 18 shows a comparison of BMP2-BMSCs cells in microcapsules with BMP-2 protein secreted by normal BMSCs (Dox 1-5 d).

FIG. 19 is a comparison of cell proliferation capacity of microvesicle cell complex cultures after rupture of the sac at various time points.

FIG. 20 is a graph showing the effect of osteoblast gene expression in microvesicle complexes.

FIG. 21 is a graph showing the effect of alkaline phosphatase (ALP) activity.

FIG. 22 shows alkaline phosphatase (ALP) staining.

Figure 23 is alizarin red staining.

Fig. 24 is a graph of promotion of mineral salt deposition following osteogenic induction (Von kossa staining).

Detailed Description

The present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the present invention are commercially available.

The technical solutions and effects of the present invention are explained in detail below in the form of examples, but the present invention is not limited to the following examples.

Example 1:

1. constructing an hBMP-2 lentiviral vector carrying a controllable Tet-on system:

designing a target gene hBMP-2 primer, directionally cloning the target gene hBMP-2 to a GV347 vector carrying a Tet-on regulation system after PCR amplification and purification, and identifying whether a product successfully constructs a hBMP-2-GV347 plasmid or not through electrophoresis and sequencing after enzyme digestion:

1.1 acquisition of hBMP-2 Gene

1) Primer synthesis: the sequence of hBMP-2 is searched in GenBank, the Primer sequence is designed by using Primer Premier5.0 software, and the Primer sequence is handed to Shanghai Jikai biology, Inc. for synthesis.

Description of the primers: contains exchange pairing basic group, enzyme cutting site and target gene 5' end sequence for PCR fishing target gene.

2) hBMP-2 gene PCR amplification and detection

PCR reaction system

The PCR reaction conditions are as follows:

3) identification of PCR products by agarose gel electrophoresis

Preparing a solution electrophoresis buffer TBE: tris (108 g); 55g of boric acid; 0.5M EDTA (pH8.0)4ml, 800ml DEPC. H was added₂And O, heating to dissolve, and fixing the volume to 1000 ml.

Ethidium Bromide (EB): 1g of ethidium bromide; DEPC. H₂O 100ml

Stirring until completely dissolved.

② preparing 100ml of 2 percent gel, weighing 2g of agarose into a triangular flask, adding 100ml of 0.5 XTBE solution, heating for 2-5min by a microwave oven to completely dissolve the agarose (paying attention to no bumping), placing the mixture at room temperature and the like to be cooled to 60 ℃, adding EB solution with the final concentration of 0.5 mug/ml, fully mixing the mixture and pouring the mixture into a plate (paying attention to gas removal).

③ when the sample is added and the sample is loaded by electrophoresis, 10 mul of PCR reaction solution is generally taken, 3 mul of bromophenol blue solution is added, the mixture is fully mixed, and the mixture is added into a gel sample adding hole, and the bubbles in the air are removed. The electrophoresis working solution is 0.5 times TBE solution, the power supply is switched on, and the sample moves from the negative electrode to the positive electrode. Electrophoresis was carried out at a constant voltage of 60-100V for about 45 minutes.

And detecting the gel plate, wherein the DNA product and the EB form an orange-yellow fluorescent compound. And (4) observing whether orange-yellow fluorescent bands appear in each lane after electrophoresis, and judging whether the sizes of the amplified fragments and the designed fragments are consistent with those of the marker.

The result of agarose gel electrophoresis after primer PCR is shown in figure 2, the left side is PCR product with the size of 1233bp which is consistent with the size of hBMP-2, and the right Marker (250bp DNA ladder Marker) is sequentially from top to bottom: 5Kb, 3Kb, 2Kb, 1.5Kb, 1Kb, 750bp, 500bp, 250bp and 100bp, which indicates that the hBMP-2 gene is successfully constructed.

4) PCR product purification

Column equilibration step: the adsorption column CA3 was placed in the collection tube, 500. mu.l of the equilibration solution BL was added, centrifugation was carried out at 12000rpm for 1 minute, the waste liquid in the collection tube was discarded, and the adsorption column was replaced in the collection tube.

Under UV light, the DNA bands of interest were cut from the agarose gel, placed in a clean centrifuge tube, and weighed. Adding 3 times of the sol solution PN into the gel block, placing in a water bath at 50 ℃ for 10 minutes, and gently shaking until the gel block is completely dissolved. Cooling the gel solution to room temperature, adding into adsorption column CA3, standing at room temperature for 2min, centrifuging at 12,000rpm for 30-60s, removing waste liquid from the collection tube, and placing adsorption column CA3 into the collection tube.

Adding 600 μ l rinsing solution PW into adsorption column CA3, centrifuging at 12,000rpm for 30-60s, pouring off waste liquid in the collection tube, and placing adsorption column CA3 into the collection tube. This step is repeated. CA3 was returned to the collection tube, centrifuged at 12,000rpm (. about.13,400 Xg) for 2min, the rinse removed, and allowed to air dry thoroughly at room temperature. Placing the adsorption column CA3 into a clean centrifuge tube, suspending and dropwise adding appropriate amount of elution buffer EB into the middle position of the adsorption membrane, and standing at room temperature for 2 min. The DNA solution was collected by centrifugation at 12,000rpm for 2 minutes.

1.2 GV347 vector cleavage

1) The enzyme digestion reaction conditions are as follows: and (3) reacting the reaction system at 37 ℃ for 2 hours, carrying out agarose gel electrophoresis on the enzyme digestion product, carrying out the same experimental steps as the experiment 1.1, and taking out the gel to observe the result in a gel imaging system.

2) Enzyme digestion reaction system

3) After gel electrophoresis, the digested GV347 plasmid was recovered and dissolved in 50. mu. L d according to 1.3 stepsd H₂In O for spare

1.3 construction of hBMP-2-GV347 recombinant plasmid

The purified PCR product was exchanged into the linearized GV347 vector

1) Reaction grouping: the purified fragment added in the experimental group is hBMP-2DNA obtained in experiment 1.3

Fragment, positive control group added internal control GAPDH, negative control group not added any post-purification PCR fragment.

2) Reaction system:

3) reaction conditions are as follows: after the reaction systems are mixed, the mixture is reacted for 2 hours at 25 ℃; the product after reaction is placed at-20 ℃ for standby

PCR identification and sequencing of hBMP-2-GV347 plasmid

1) The enzyme cutting site of the GV347 vector is subjected to double enzyme cutting by Age I to obtain a linearized vector, the linearized vector is hBMP-2-GV347 plasmid after being linked with a target gene PCR product, whether positive cloning is performed or not can be detected by adopting a PCR amplification detection method, primers on the vector and a primer PCR of the target gene are used for identification, an experimental group (positive cloning) and a negative control group (no-load self-connection) are set, and the third group is a positive control Group (GAPDH) for comparison.

2) Dipping the transformant, adding 20 mu l of LB liquid culture medium containing ampicillin (100 mu g/ml), blowing, uniformly mixing, taking 1ul as a template, and detecting and identifying whether the target gene hBMP-2 is directionally connected or not through PCR amplification.

3) Description of the primers: KL9288-P3 was located on the target gene, pEGFP-N-3GV347 vector, and was used for PCR identification of the groups of transformants in Experimental step 3

3) PCR reaction system

4) And (3) PCR reaction conditions:

5) agarose gel electrophoresis of PCR products of experimental group, positive control group and negative control group, and 1 st hole negative blank group (ddH)₂O), well 2 is a negative control (no-load self-ligation), the third is a positive control (GAPDH), well 4 is a marker, and wells 5 to 12 are experimental. The PCR products of the experimental group were sent to Gjkey company for sequencing.

2. Preparing competent E.coli DH5a, transforming and amplifying hBMP-2-GV347 plasmid, and co-infecting 293T cell with helper plasmid; simultaneously co-infecting 293T cells with unloaded GV347 and helper plasmid; collecting and concentrating the slow virus liquid, and determining the titer of the two virus liquids by a drug sieving method; the two lentivirus vectors are respectively transfected into 293T cells, and the expression condition of a target gene in each group of cells is determined by using western-blot with or without DOX induction.

Cleavage of GV347 vector

The agarose gel electrophoresis of the GV347 vector after Age I cleavage is shown in FIG. 3. 10kb Marker (1kp DNA ladder Marker, 10kb, 8kb, 6kb, 5kb, 4kb, 3.5kb, 3kb, 2.5kb, 2kb, 1.5kb, 1kb, 750bp, 500bp, 250bp from top to bottom) 2#, and 3#, non-digested vector.

The hBMP-2 recombinant plasmid can stabilize the EPBMP-2 protein in 293T cells after being transfected by lentivirus.

(1) Extraction of hBMP-2-GV347 plasmid

1) Adding a small amount of E.coli DH5a bacterial liquid of an experimental group of transformed recombinant plasmid hBMP-2-GV347 into 4ml of resistant LB culture medium, culturing at 37 ℃ for 16 hours, diluting the bacterial liquid into 200ml of resistant LB culture medium according to the proportion of 1:100, and culturing at 37 ℃ for 16 hours; centrifuging at 6000rmp/min for 15min at 4 deg.C, completely removing supernatant, and harvesting bacteria;

2) 5ml Buffer I was added and the pellet was fully suspended and shaken. The bacterial suspension was transferred into a 50ml centrifuge tube. 5ml Buffer II was added, the tube was inverted gently and left at room temperature for 5min to allow complete lysis of the bacteria. 5ml Buffer III was added and mixed well until white floc was produced. And standing on ice for 10-15 min.

3) The lysate was centrifuged at 12,000rpm at 4 ℃ for 15min, the supernatant carefully aspirated and transferred to a new 50ml centrifuge tube. Add 10ml of isopropanol, invert the tube and mix well. And standing on ice for 10-15 min. The mixture was centrifuged at 4 ℃ at 12,000rpm for 10min, the supernatant was discarded, and 0.5ml of Buffer I was added to completely dissolve the pellet. Transferring the mixture into a new 1.5ml centrifuge tube, and standing the centrifuge tube for 10-20min at room temperature.

4) After the plasmid crude extract was centrifuged at room temperature for 2min at high speed, the supernatant was transferred to a new 1.5ml centrifuge tube. To 0.5ml of the crude plasmid extract was added 100. mu.L of Buffer IV (impurity-removed solution A), and the mixture was centrifuged at 12,000rpm for 2min to obtain the supernatant. This step is repeated. Add 70. mu.L of Buffer V (impurity-removed solution B), centrifuge at 12,000rpm for 5min, and collect the supernatant.

5) Adding 0.5ml isopropanol, mixing well, standing at room temperature for 10 min. Centrifuging at 12,000rpm at room temperature for 10min, discarding the supernatant, washing with 1ml of 70% ethanol, and air drying at room temperature for 5 min.

6) Dissolving the precipitate in 0.5ml of TE, adding 200 mu L of Buffer VI (impurity removing solution C), uniformly mixing, standing on ice for 10-30 min, centrifuging at 12,000rpm at room temperature for 10min, discarding the supernatant, washing twice with 1ml of 70% ethanol, and inversely drying at room temperature for 5-10 min. Dissolving the precipitate with 500 μ L of sterilized ultrapure water, and storing at-20 deg.C.

(2) Lentiviral packaging plasmids and viral titer detection (FIG. 1 flow sheet)

293T cell transfected by recombinant plasmid

24 hours before transfection, 293T cells at logarithmic growth phase were trypsinized to adjust cell density to 1.2X 10⁷The cells/20 ml were inoculated in a six-well plate and cultured in a 5% CO2 incubator at 37 ℃. Can be used for transfection when the cell density reaches 70-80%. Cell culture medium was changed to serum-free medium 2 hours before transfection.

20. mu.g of the prepared recombinant GV347-hBMP-2 vector, 15. mu.g of the pHelper 1.0 vector, and 10. mu.g of the pHelper 2.0 vector were mixed with the corresponding volume of Opti-MEM, and the total volume was adjusted to 2.5ml, and the mixture was cooled at room temperatureIncubate for 5 min. Mu.l Lipofectamine 2000 reagent was mixed with 2.4ml Opti-MEM and incubated for 5min at room temperature. The diluted DNA was mixed with the diluted Lipofectamine 2000 without shaking. Incubate at room temperature for 20min to form a transfection complex of DNA with Lipofectamine 2000 dilution. The complex is transferred to 293T cell culture solution and mixed evenly, and the temperature is 37 ℃, and the CO content is 5 percent₂After 8h incubation in the cell incubator, the medium containing the transfection mixture was removed and the remaining transfection mixture was washed gently by adding 20ml of PBS.

To each flask of the cells, 25ml of a cell culture medium containing 10% fetal bovine serum was added, and the cells were cultured in a 5% CO2 cell culture chamber at 37 ℃ for another 48 hours. Preparing a negative virus: the transfection procedure was repeated by adding empty GV347 negative plasmid to 293T cells.

Harvesting and concentration of viruses

After 48h of culture, collecting the supernatant of the transfected 293T cells; centrifuging at 4 deg.C and 4000rmp for 10 min; the supernatant was filtered. Adding 20% sucrose solution into the bottom of the centrifuge tube, centrifuging at 4 deg.C and 10000rmp for 2 h. Removing supernatant, adding 200 μ l of Opti-MEM into each tube to dissolve precipitate; placing in a refrigerator at 4 deg.C for 2h, and shaking for 1 time every 20 min; centrifuging at room temperature at 500rmp for 1min, concentrating virus solution, filtering with 0.22um filter tip, and freezing at-80 deg.C. Marking is done to distinguish the negative virus liquid without hBMP-2 from the positive virus liquid with hBMP-2.

Tet-on regulation system detection of 293T cell infected by virus

293T cells in logarithmic growth phase were digested with 0.25% trypsin-0.125% EDTA to prepare a cell suspension (5X 10)³One/ml) were inoculated in 24-well plates at 37 ℃ with 5% CO₂Culturing until the cell fusion degree reaches about 20%. Positive virus cells transfected 293T cells: after the plate was washed with PBS, 20. mu.l of positive virus was added to each well, and serum-free DMEM was supplemented to 500. mu.l, and polybrene was added thereto to maintain the concentration at 8 ug/ml. After 12h, removing the virus mixed solution, and adding 500 mu l of DMEM containing 10% FBS into each well; 5 μ g DOX was added simultaneously. After 48 hours, the image was taken under a fluorescent microscope to observe the expression of fluorescence. The negative virus was repeated according to this procedure.

Method for determining positive and negative virus titer by drug screening method

1 day before assay, 96-well plates were plated with 4X 10 cells per well for titer determination⁴One cell per 100. mu.l of 293T cells. 10 sterile EP tubes were prepared. 90. mu.l of serum-free medium was added to each tube. And adding 10 mul of positive virus stock solution to be measured into the 1 st tube, slightly shaking and uniformly mixing, and adding 10 mul into the 2 nd tube. The same procedure as above was continued until the last tube, corresponding to 10-fold dilution of each tube compared to the previous one. The 1 st tube is labeled 1E + 1. mu.l, the 2 nd tube diluted 10 times is labeled 1E + 0. mu.l, the 3 rd tube is labeled 1E-1. mu.l, and so on, and the 10 th tube is 1E-8. mu.l.

And selecting cell wells with good growth states, marking each tube corresponding to each cell well, and removing 90 mu l of culture medium from each well. Respectively adding 90 mul of corresponding diluted virus solution, placing the virus solution into an incubator for 12 hours, changing the solution, adding 100 mul of complete culture medium into each hole, simultaneously adding 1 mug DOX, and opening a Tet-on switch. Changing the solution within 48 hours, and continuously keeping the DOX concentration at 10 mu g/ml; puromycin was added to maintain the concentration at 5 μ g/ml. The culture was continued for 2 days, and the survival was microscopically.

Determining the virus titer, and calculating the formula as follows: the number of viable cells in the group with the minimum concentration/the group concentration

The virus titer of the negative virus was determined by the same procedure.

(3) Western-blot detection of BMP-2-GV347 lentivirus vector expression BMP-2 expression

Preparation: 10% SDS solution, 0% Ammonium Persulfate (APS) solution, 0.5mol/L Tris & HCl (pH 6.8) solution, electrophoresis buffer (Tris 3.03g, glycine 18.77g and SDS 1.0g, distilled water 1000ml), transfer buffer (glycine 2.9g, Tris 5.8g and SDS 0.37g, methanol 200ml, distilled water), TBS buffer, TBST buffer, blocking solution: weighing 5g of skimmed milk powder, dissolving in 100ml of TBST buffer solution, storing at 4 deg.C, and returning to room temperature for use.

Extraction of cellular proteins

After the positive virus transfects the 293T cells of the 24-well plate, the solution is changed for 12 hours, DOX is added, the concentration is maintained at 10 mu g/ml, the culture is carried out for 96 hours, and PBS is washed for 2 times. Adding 50 μ l of 1 × lysine Buffer (protein electrophoresis loading Buffer), blowing to fully crack the cells, and cracking the cells for 10-15 min on ice; centrifuging at 4 deg.C and 12000rpm for 5min, and water-bathing in boiling water for 10 min; centrifuging at 12000rpm at 4 deg.C for 1min to obtain positive virus sample solution, labeling as OE (dox +) group, and storing at-80 deg.C for use.

Ordinary 293K cells, negative virus transfected cells without DOX induction, negative virus transfected cells with DOX induction and positive virus transfected cells without DOX induction are extracted from each group according to the steps, and the samples are respectively marked as con group, NC (DOX-) group, NC (DOX +) group and OE (DOX-) group

SDS-PAGE electrophoresis

1) Preparing glue: cleaned and fixed on a rack, a 10% release gel prepared according to table 1 below was poured between the glass plates, the surface was sealed with ultrapure water, and polymerized at room temperature. When a distinct interface between the water in the upper layer and the separation gel layer appeared, indicating that the polymerization of the separation gel was complete, it was blotted dry with absorbent paper and then filled with 5% concentrated gel (formulated according to table 2).

TABLE 110 ml 10% separation gel formulation

TABLE 25ml 5% concentrated gum formulation

2) Loading: after the gel solidified, the comb was removed, the loading wells were washed with running buffer, 20ul samples were taken from each group, and SURVIVIN-3FLAG-GFP (molecular size 48KD) was added as a positive sample control.

Loaded onto the gel.

3) Electrophoresis: adding electrophoresis buffer solution into the electrophoresis tank, carrying out electrophoresis at constant voltage of 80V, and continuously running gel for 2 hours.

The clones obtained after transformation were randomly selected 8 clones (5-12 groups) and identified by PCR with the control group, and the results of electrophoresis are shown in FIG. 4.

FIG. 5 is a diagram illustrating: 1#: negative blank (ddH 2O); 2#: negative control (no-load self-ligation control group); 3#: positive control (GAPDH), 4 #: marker (1kp DNA ladder Marker) is 10Kb, 8Kb, 6Kb, 5Kb, 3Kb, 1.5Kb, 1Kb, 500bp, 100bp, 5# -12# from top to bottom: transformant No. BMP 21-8. The experimental group showed 1382bp fragment, and the negative blank group and the negative control group showed no band. Thus indicating that the construction of positive clone is successful.

4) Immunoblotting (wet transfer) by preparing 1L of a transfer buffer and cooling to 4 ℃; after electrophoresis is finished, slightly washing the gel by using 1 × transfer buffer solution, wetting a nitrocellulose membrane Hybond-C by using deionized water, and transferring the nitrocellulose membrane Hybond-C and the electrophoresis gel into the transfer buffer solution for balancing for 15 minutes; cutting 4 Whatman3mm filter papers with the same size as the glue, and placing the filter papers in a transfer buffer solution for balancing; sequentially placing sponge, filter paper, gel, PVDF membrane, filter paper and sponge according to a sandwich method to remove all bubbles, placing the PVDF membrane on the anode side, placing the gel on the cathode side, inserting into an electrophoresis tank, pouring into membrane transfer buffer solution, performing electric transfer for 150 minutes under the condition of 300mA constant current, and transferring protein onto the PVDF membrane

5) And (3) combining an ECL method with an x-ray film for developing and sealing: sealing the PVDF membrane by using a sealing solution at room temperature for 1 hour or overnight at 4 ℃; primary antibody incubation: the blocked PVDF membrane was removed, immersed in 1 XTSST buffer, washed slowly on a shaker for 5 minutes, TBST at 1: primary anti-FLAG was diluted at 2000, and the PVDF membrane was blocked and incubated at room temperature for 2 hours overnight at 4 ℃. Washing the membrane: washing the membrane with TBST for 3 times, 10 minutes each time; and (3) secondary antibody incubation: blocking solution diluted secondary antibody mouse IgG (1: 2000), immersing the membrane in the secondary antibody diluent, and incubating the PVDF membrane by gentle shaking at room temperature for 2 hours; the membranes were washed 3 times 10 minutes each with TBST.

Developing with ECL + plusTM Western blotting system kit from Amersham; placing the PVDF film on a flat plastic wrap, 1: mixing the solution A and the solution B in a ratio of 40 to obtain a mixed solution, uniformly dropwise adding the mixed solution on a PVDF membrane, and reacting for 5 minutes in a dark place; taking out the film, removing the redundant ECL substrate reaction liquid, putting the film into a cassette, laying a preservative film (avoiding generating bubbles), putting an X-ray film (avoiding the movement of the X-ray film), closing the cassette, and exposing for 1-2 minutes; taking out the X-ray film, putting the X-ray film into the developing solution, taking out the X-ray film after about 1 minute, rinsing the X-ray film in clear water for a few seconds, then putting the X-ray film into the fixing solution for 5 minutes, then taking out the X-ray film, airing the X-ray film, and observing the result.

3. Respectively transfecting juvenile male rat BMSCs with hBMP-2-GV347 lentivirus and unloaded GV347 lentivirus vectors to obtain Tet-on/hBMP-2 positive cells and negative cells, and screening the optimal cell transfection Multiplicity (MOI) and the optimal screening concentration of puromycin (Puro). Comparing the proliferation capacities of the cells of each group before and after transfection by using a CCK8 method and WST-8 respectively under the condition of or without DOX induction after transfection; detecting BMP-2, RUNX2 and OCN gene expression in the transfected MSCs by real-time fluorescent quantitative PCR; the expression of BMP-2 protein in the cells is verified by western-blot; enzyme linked immunosorbent assay kit (ELISA) detects the expression of BMP-2 protein in extracellular fluid. After the transfection of MSCs, the activity of alkaline phosphatase (ALP) is detected, and alizarin red and Von Kossa staining are carried out to observe the osteogenic differentiation condition of the cells.

3.1 hBMP-2-GV347 lentivirus transfection of SD rat BMSCs and concentration screening

Taking 6 th generation BMSCs with good growth state, digesting and counting, and then taking 1 × 10⁶The BMSCs (Becton Dickinson) were inoculated on a 10cm dish, 10ml of L-DMEM (containing 10% FBS) was added thereto, and 5% CO was added at 37 ℃₂The incubator was overnight. The culture medium was aspirated off the dish, 5ml of L-DMEM without FBS was added, and at the same time, Lentiviral-BMP2 and Lentiviral-GFP lentivirus solutions at multiplicity of infection of 10,30 and 50 were added to the culture medium, respectively, and 8. mu.g/ml Polybrene was added to increase the transfection efficiency, and the cells were transferred to 37 ℃ in 5% CO₂And continuing culturing in the incubator. After 20 hours of transfection, virus and culture medium were aspirated, and L-DMEM was added to continue the culture. Cell fluorescence was observed under a fluorescence microscope 72h after lentivirus transfection and transfection efficiency was calculated. The calculation method is as follows: the number of green fluorescent expressing cells in the total number of cells was counted in 6 fields of 40-fold random per well, and the infection efficiency per MOI value was the average infection efficiency of 6 fields. When the cells grow to 80-90%, the cells are subcultured and expanded for later use, and redundant positive virus cell strains and negative virus cell strains are frozen and stored.

Puromycin screening positive and negative virus strain cell

1) Collecting 5 th generation BMSCs with good growth state, digesting, and repeatingSuspending cells at a cell density of 5X 10⁴The cells were inoculated in a six-well plate and the complete medium (1 ml, containing 10% fetal bovine serum L-DMEM) was cultured in a 5% CO2 incubator at 37 ℃.

2) When the cell fusion degree is less than 25%, adding complete culture medium and puromycin into each hole, setting puromycin gradient concentration to be 1.5 mu g/ml, 2 mu g/ml, 2.5 mu g/ml, 3 mu g/ml and 3.5 mu g/ml, respectively, changing liquid every 72 hours for 3 holes of each group, and observing the cell growth condition.

3) After 7 days, the minimum concentration at which all normal BMSCs were killed was the ideal puromycin screening concentration.

4) Resuscitating cryopreserved positive and negative virus strains: taking out the freezing tube from the liquid nitrogen tank, rapidly placing into 37 deg.C water bath, shaking, completely thawing within 1min, adding appropriate amount of culture solution, inoculating into T15 cell culture bottle with inoculation concentration of 1 × 10⁶And L, placing the mixture in a 37 ℃ incubator for static culture, replacing the culture solution the next day, continuing the culture, and observing the growth condition.

5) And (3) screening positive virus strains and negative virus strains in a 96-well plate by using an ideal puromycin screening concentration according to an ideal concentration, adding 10 mu g/ml Dox to induce and open Tet-on, observing the condition of each clone in the plate under the excitation of blue light of a fluorescence microscope, continuously screening for 7 days, picking out a monoclonal with EGFP reaching 100%, and culturing in a 6-well plate to obtain cells (positive cells) containing hBMP-2-GV347-BMSCs and cells (negative cells) containing no-load GV 347-BMSCs.

CCK8 method for determining change of BMSCs proliferation capacity after hBMP-2-GV347 lentivirus vector transfection

Digesting transfected BMSCs with 0.25% trypsin-0.02% EDTA, resuspending with L-DMEM after centrifugation, at 1X 10⁴Each well was seeded in a 96-well plate and divided into 5 groups of three experimental wells (n ═ 3): positive virus group (adding DOX 10ug/ml to induce open Tet-on), positive control group (not adding DOX induction), negative virus group (adding DOX 10ug/ml to induce open Tet-on), normal cell group (BMSCs without lentivirus transfection), blank control group (no cell, only containing L-DMEM), and culturing in a thermostat at 37 deg.C and 5% CO 2. After 24 hours, the medium volume was adjusted to 100. mu.l per wellAfter adding 10. mu.l of CCK8 reagent and incubating at 37 ℃ and 5% CO2 for 2 hours, the optical absorption (OD) of each well was measured at 450nm and the results were recorded.

After the solution in the wells is removed, PBS is washed for 3 times, L-DMEM is added for continuous culture, and DOX induction is continuously added to the positive virus group and the negative virus group. OD values were further measured at 1 day, 3 days, 5 days, 7 days, and 10 days after the inoculation, and the change in proliferation potency was compared among the groups.

q-PCR detection of BMSCs BMP-2 mRNA expression after gene transfection and osteogenic genes RUNX2, OCN expression

Designing and synthesizing a primer: searching a target gene mRNA sequence on GenBank, and designing a specific primer in a CDS region:

1) sequence Name H-BMP-2 (amplified fragment length 72bp)

Forward Primer:5’-AAAACGTCAAGCCAAACACAA A-3’

Reverse Primer:5’-TCCACGTACAAAGGGTGTCTCTT-3’

2) Sequence Name H-beta actin (amplified fragment length 106bp) reference gene

Forward primer:5’-GCATGGGTCAGAAGGATTCCT-3’

Reverse Primer:5’-TCGTCCCAGTTGGTGACGAT-3’

3) Sequence Name R-OC (osteopecalin) (length of amplified fragment: 101bp)

Forward Primer:5’-CTCACTCTGCTGGCCCTGAC-3’

Reverse Primer:5’-CCCTCCTGCTTGGACATGAA-3’

4) Sequence Name: R-RUNX2 (length of amplified fragment: 105bp)

Forward primer:5’-AGGCACAGACAGAAGCTTGATG-3’

Reverse primer:5’-AGGCGGGACACCTACTCTCA-3’

5) Sequence Name: R-GAPDH (amplified fragment length: 110bp) internal reference gene

Forward Primer:5’-AGGGCTGCCTTCTCTTGTGA-3’

Reverse Primer:5’-AACTTGCCGTGGGTAGAGTCA-3’

The RNA extraction and PCR detection steps were as described above.

Western blot method for detecting BMP-2 protein secreted by transfected cells

The protein extraction and electrophoresis detection steps are as described above.

Detection of BMP-2 expression by ELISA

Grouping experiments: 1) positive cell group: DOX (1 mu g/mL) is added into BMP2-BMSCs cells for induction;

2) positive control group: BMP2-BMSCs cells were not DOX added;

3) negative cell group: GFP-BMSCs are induced by adding DOX (1 mu g/mL);

4) blank control group: BMSCs not transfected with lentivirus.

Collecting cell supernatants at day 1, day 2, day 3, day 5, day 7, day 10 and day 14, counting cells, collecting 5 tubes (n is 5) in each group, and measuring BMP-2 protein secretion of the supernatant in each group by ELISA method, which comprises the following steps:

1) adding cell supernatant to be detected and standard substances (100 mu L/hole) with different concentrations into the reaction hole, and fully and uniformly mixing; 2) incubating for 45min at 37 ℃, fully washing for 6-8 times, and printing on filter paper;

3) add antibody working solution (50. mu.L/well) to each well, mix well, incubate at 37 ℃ for 25min, wash well 5 times.

4) Adding 100 μ L of enzyme-labeled antibody working solution into each well, incubating at 37 deg.C for 10-12min, and washing for 5 times.

5) Adding 100 mu L of substrate working solution into each hole, and incubating for 20min at 37 ℃ in a dark place;

6) adding 100 mu L/hole of stop solution, and fully and uniformly mixing;

7) measuring OD450 values on a microplate reader, taking standard substances of 2000, 1000, 500, 250, 125, 62.5, 31.2 and 0pg/mL as abscissa and absorbance value as ordinate, and drawing a standard curve by using a drawing software;

8) calculating the concentration of BMP2 in the sample to be tested

Osteogenic differentiation capacity of BMSCs transfected by BMP-2 gene

Alkaline phosphatase (ALP) Activity assay

BMP2-BMSCs were transfected and cultured in 6-well plates, L-DMEM and DOX were added at 1. mu.g/mL, and ALP-transfected cell activity was examined after 7d and 14d transfection in the following manner. Normal BMSCs and GFP-BMSCs were also set up as blank and parallel controls.

(1) Washing with PBS for 3-4 times (3 min/time);

(2) 0.1% Triton X-100 for 20 minutes; transferring the solution to a new EP tube;

(3) centrifuging at 14000rpm/min for 20min at 4 deg.C;

(4) the supernatant was discarded, the pellet was transferred to a new EP tube, an ALP reaction substrate (20. mu.L of p-nitrophenyl 5m M phosphate, 20. mu.L of 5mM Mg Cl2 and 100. mu.L of 1M Tris-HCl) was added, and incubation at 37 ℃ for 30 minutes in an incubator;

(5) adding NaOH to terminate the reaction, and detecting the absorbance value at 410nm by using an enzyme-labeling instrument;

(6) the protein content in the corresponding samples was determined using the BCA method, and the activities of alkaline phosphatase in each group were compared by dividing the absorbance by the protein content.

Calcium nodule detection after osteogenic differentiation: conventional alizarin red staining Von Kossa staining, and observing and photographing under an optical microscope

4. Preparing microcapsules with a specific size by coaxially copolymerizing microfluid using a newly synthesized high molecular material, namely, carboxymethyl cellulose containing a phenolic group (CMC-Ph); meanwhile, rat MSC cells are wrapped to probe the biological performance of the microcapsule; the property and cell survival rate of CMC-Ph and hBMP-2-GV347-BMSCs after forming micro encapsulation are discussed.

The conditions of the high-voltage electrostatic field capsule forming device are 4.0 multiplied by 10³V, the needle point is 25mm away from the liquid surface, the advancing speed of the injector is 30mm/h, and the sodium alginate (APA) microcapsule cell complex is prepared. Preparation of CMC-Ph cell microcapsule complex: the novel microcapsules are constructed by using carboxymethyl cellulose (CMC-Ph) containing phenol group and coaxial copolymerization microfluid, the overall shape and size of the microcapsules are observed, and various parameters are adjusted to prepare the microcapsules with monodisperse property with the diameter of 80-160 mu m. Fluid velocity in the microfluidic device is 50 μ l/min; the external fluid is 8 ml/min. The particle sizes of the two groups of microcapsule complexes were measured and the standard deviation was calculated. The two groups of microcapsules are respectively centrifuged for 5min at the speed of 1000rpm/min, 2000rpm/min, 5000rpm/min and 10000rpm/min, and the integrity rates of the microcapsules are compared. Two groups of microcapsules respectively wrap gene transfected cells and non-transfected cellsThen, using Live/Dead viability kit to detect the survival rate of the cells in the microcapsule; and measuring the in vivo cell survival rate of the APA microcapsule complex by an FDA/EB staining method. The physical properties of the two groups of microcapsules were compared with biocompatibility.

Detecting the proliferation condition of cells in the microcapsules by using a CCK8 kit at each time point, and drawing a growth curve to evaluate the growth and metabolism condition of the cells; enzyme-linked immunosorbent assay is used for detecting BMP-2 protein secreted by extracapsular cells after DOX action and corresponding secretion kinetic analysis is carried out.

Preparation of solutions

1) Preparing a liquid paraffin mixed solution: 2.5ml of hydrogen peroxide is dissolved in 500ml of liquid paraffin, the mouth of the beaker is sealed by a film and is protected from light in the whole process, the beaker is magnetically stirred for 14 hours at the temperature of 25 ℃, and the beaker is centrifuged for 10 minutes at the speed of 2500 rpm; discarding unbound H₂O₂(ii) a Adding 5 wt% lecithin, magnetically stirring at 25 deg.C for 24 hr, mixing, and storing in dark (1-2 days).

2) Configuration of the internal fluid: shearing sterile CMC-Ph into fine shape, and dissolving in 1.0 wt% of water solution; fully dissolving the mixture into colorless state by magnetic stirring; horse Radish Peroxidase (HRP)100units/mL was added just before the experiment.

Experimental methods

Preparation of CMC-Ph (phenolic carboxymethyl cellulose-containing) microcapsules

Using a liquid copolyfluidic device, and a micro-syringe pump as a generator, with liquid paraffin (containing H)₂O₂And lecithin) as a peripheral liquid solution, i.e., a continuous phase (external fluid), a carboxymethyl cellulose solution containing a phenol group (containing HRP: horseradish peroxidase, > 100units/mL) as the dispersed phase (inner fluid) extruded from the superfine pipe (the continuous phase and the dispersed phase are prepared in advance, see the preparation of the liquid in this chapter). Standing the collected microcapsule for 20-30min, removing liquid paraffin after the microcapsule is completely deposited at the bottom of the collecting tube, adding PBS solution, and centrifuging and washing for 2 times at 2000rpm/min × 5min in a centrifuge to obtain purer microcapsule. Observing the microcapsule shape under microscope and continuously adjusting various parameters to meet the requirement of the particle size (80-160 μm) of the microcapsule.

Fig. 12 is a graph showing the relationship between the microcapsule size and the flow rate of liquid paraffin. It shows that the diameter of the microcapsule becomes smaller and smaller as the flow rate of the liquid paraffin is increased.

Microencapsulated gene-transfected BMSCs

BMP2-BMSCs, GFP-BMSCs and blank untransfected BMSCs were selected and used as the selection samples, each of which was 1X 10⁷Per mL, digesting the cell suspension by a conventional method for later use; dispersed phase: 0.1g CMC-Ph was dissolved in 4ml L-DMEM (containing 10% FBS) and magnetically stirred for 30min until completely dissolved; adding HRP (horse radish peroxidase) of 1mg/mL, and fully dissolving; adding BMSCs, and fully and uniformly mixing to avoid excessive stirring; adjusting the flow rate on the micro pump: continuous phase (liquid paraffin with lecithin): 16 mL/min; dispersed phase: 50 μ L/min.

Collecting microspheres by a centrifugal tube, and standing for 10-20 min; centrifuging at 1100rpm/min for 2min, and sucking off the paraffin at the upper layer; adding 10mL of PBS into each tube for washing, then centrifuging at 1100rpm/min for 2min, and discarding the supernatant; repeatedly washing for 3 times; adding complete culture medium, blowing gently to make the microcapsule fall off, and transferring into culture plate; placing at 37 deg.C, keeping constant temperature, and adding 5% C0₂Culturing in an incubator. Changing the solution 1 time every 2-3 days. FIG. 13 is an under-the-mirror image of microencapsulated cells.

Detecting viability of cells within microcapsules

Preparing Live/Dead vitality/cytotoxin Kit staining solution, namely adding 2 mu L of solution B into 1ml of PBS and uniformly mixing; then adding 0.5 mu L of solution A, fully and uniformly mixing, and storing in a dark place with the effective period of 24 hours; selecting CMC-Ph microcapsule complex cultured for 1 day, 7 days, 14 days, 21 days, 28 days and 35 days, adding dyeing working solution 200 μ L into each hole, and reacting at room temperature in dark and room temperature for 30-45 min; 37 ℃ and 5% CO₂Culturing in incubator for 30min, washing with PBS for 2-3 times, each for 2 min; cell survival was observed under fluorescent microscope: each sample was photographed at random with 6 different fields, green fluorescence was live cells and red fluorescence was dead cells. The survival rate is calculated. FIG. 14 is a dead and live staining of cells within microcapsules after 24 hours of culture. Green for live cells and red for dead cells.

Detection of cell proliferation curves within microcapsules

On days 1, 4, 7, 10, 14 and 17 after the formation of the microvesicle cell complex,detecting CCK-8 on day 21; 200ul of culture medium and cell-coated microcapsules are added into each well of a 24-well plate; adding 20 mu L of CCK-8 solvent; 37 ℃ and 5% CO₂Culturing for 2h in an incubator; the microplate reader measures the spectrophotometric value at a wavelength of 450 nm. FIG. 15 shows the proliferation of cells in the microcapsules after 2 weeks of culture.

FIG. 16 shows the proliferation of cells within the microcapsules. It was shown that within 1-7 days, BMSCs proliferated more actively, reached a maximum, and then gradually fell back.

Quantitative detection and kinetic analysis of BMP-2 protein secreted by extracellular cells of microcapsules

Quantitative determination of BMP-2 protein: collecting the culture medium in a 6-well plate at each time point of culturing the microcapsule + BMP2/Tet-on BMSCs complex; adding DOX into the positive group, acting for at least 24 hours, and collecting the supernatant for determination; directly collecting supernatant in a microcapsule + non-transfected BMSCs group; centrifuging at 4000rpm/min for 10 minutes at 4 ℃; the precipitate was discarded, and the supernatant was aspirated to determine the protein content of BMP-2 using an ELISA kit.

Kinetic analysis of the secretion of BMP-2 protein by the Microcystis Complex: adding DOX1 μ g/mL into the microcapsule cell complex BMP-2-Tet-On-BMSCs component for 5 days and 28 days respectively; collecting the culture medium in the 6-well plate; centrifuging at 4000rpm/min at 4 deg.C for 10 min; the supernatant was aspirated and the BMP-2 protein content was determined separately using an ELISA kit.

Respectively coating BMP2-BMSCs, GFP-BMSCs and BMSCs with the CMC-Ph microcapsule, and inducing by using osteogenic induction liquid and DOX; dissolving the microcapsules with cellulase at each time point and detecting proliferation and viability of the ruptured MSCs; detecting the expression of ALP gene, type I collagen (COLL I), bone calcium (OC) gene, bone bridge (OPN) gene and RUNX2 gene by RT-PCR; detecting ALP activity by using an alkaline phosphatase kit; calcium-cobalt staining to assess the expression of ALP; alizarin red was used simultaneously to detect calcium nodule formation and Von Kossa to detect cellular calcium salt deposition. FIG. 17 is a comparison of BMP2/Tet-on-BMSCs cell secretion in microcapsules and BMP-2 protein secretion from BMP2/Tet-on-BMSCs without microencapsulation. FIG. 18 shows a comparison of BMP2-BMSCs cells in microcapsules with BMP-2 protein secreted by normal BMSCs (Dox 1-5 d).

Effect of CMC-Ph microcapsule wrapping cells on induced osteogenesis

Grouping: (1) CMC-Ph microcapsules + BMSCs;

(2) CMC-Ph microcapsules + GFP-BMSCs;

(3) CMC-Ph microcapsule + BMP2-BMSCs

Each group was plated in 6 wells (n: 6) in parallel, and then osteogenic induction solution and doxycycline (1 μ g/mL) were added thereto, followed by culture for 7 days, 14 days, and 21 days, respectively. The microcapsules were then lysed with cellulase (5units/mL), the cells harvested by centrifugation, replanted in 6-well plates, and 2 mL/well of complete medium was added. After 3 days, each analysis was performed.

Proliferation and viability of cells after rupture of the sac

1) Collecting cells after the microcapsule rupture, centrifuging at 2000rpm/min for 2-3min, and sucking and removing supernatant;

2) transferring the cells to a new 96-well plate, and adding a fresh L-DMEM medium for culture;

3) adding CCK-810 mu L into each well of cultured 1 st, 2 nd, 3 rd, 4 th, 5 th, 6 th and 7 th wells;

4) placing 96-well plate at 37 deg.C and 5% CO₂Incubating for 3h in an incubator;

5) the absorbance of each well was measured at 450nm on a microplate reader.

Detection of osteogenic gene markers

Primer design Synthesis

Searching a target gene mRNA sequence on GenBank, and designing a specific primer in a CDS region:

the primer sequence is as follows:

1) sequence Name: R-ALP (amplified fragment length: 100bp)

Forward primer:5’-GCACGACAATCGGGATGAAC-3’

Reverse primer:5’-TCCAGCAGGACGGTCATCA-3’

2) Sequence Name R-OC (osteopecalin) (length of amplified fragment: 101bp)

Forward Primer:5’-CTCACTCTGCTGGCCCTGAC-3’

Reverse Primer:5’-CCCTCCTGCTTGGACATGAA-3’

3) Sequence Name: R-Collagen I (length of amplified fragment: 151bp)

Forward Primer:5’-CTCCCAGCGGTGGTTATGAC-3’

Reverse Primer:5’-TGCTGGCTCAGGCTCTTG A-3’

4) Sequence Name: R-OPN (amplified fragment length: 100bp)

Forward primer:5’-GCTGACGCTGGAAAGTTGGA-3’

Reverse primer:5’-TTCCTCGTCGCTTTCCTTCA-3’

5) Sequence Name: R-RUNX2 (length of amplified fragment: 105bp)

Forward primer:5’-AGGCACAGACAGAAGCTTGATG-3’

Reverse primer:5’-AGGCGGGACACCTACTCTCA-3’

6) Sequence Name: R-GAPDH (amplified fragment length: 110bp) internal reference gene

Forward Primer:5’-AGGGCTGCCTTCTCTTGTGA-3’

Reverse Primer:5’-AACTTGCCGTGGGTAGAGTCA-3’

The company for synthesizing the primers and the type of synthesizer used. Synthesis company: sangon synthesis apparatus: ABI 3900 desktop high throughput DNA synthesizer.

Extracting cell RNA:

taking BMSCs cells transfected by positive viruses in a good growth state, digesting and centrifuging, precipitating the cells into a 1.5mL EP tube, adding 1mL Trizol reagent, fully shaking and uniformly mixing, and standing at 25 ℃ for 5 min.

Sucking 0.2mL of chloroform, adding into an EP tube, covering the lid tightly, shaking with a shaker for 15s sufficiently, incubating at room temperature for 2-3min, centrifuging at 4 ℃ 12000rpm/min × 15min with a centrifuge, and transferring the supernatant to a new 1.5mL EP tube.

Adding isopropanol with the same volume as the supernatant, mixing gently, incubating at-20 deg.C for 25min, centrifuging at 4 deg.C 12000rpm/min for 10min, and removing the supernatant. The precipitate was washed once with 800. mu.L of 75% ethanol containing DEPC water, centrifuged at 7500rpm/min at 4 ℃ for 5min, and the ethanol was aspirated off. Vacuum drying for 8min (not completely drying), adding DEPC treated water 30. mu.L to dissolve RNA, and storing at-80 deg.C for use.

The reaction mixture was prepared according to the following system (using RNase I (Promega) from RNase-free), digested at 37 ℃ for 30min, and then inactivated at 65 ℃ for 10 min.

The method comprises the following steps:

1) the same volume of phenol was added, mixed by reversing the top and bottom sufficiently, centrifuged at 10,000rpm/min for 5min, and the supernatant was aspirated.

2) Chloroform of the same volume was added, and after mixing uniformly in the same manner, the mixture was centrifuged at 10,000rpm/min for 10min, and the supernatant was aspirated.

3) Adding isopropanol with the same volume, gently and completely mixing, and standing at-20 deg.C for 15 min;

4) centrifuging at 4 deg.C at 10,000rpm/min for 10min, collecting RNA precipitate, and discarding supernatant;

5) washing twice with 75% ethanol, and air drying in a super clean bench;

6) the precipitate was dissolved with 30. mu.L of DEPC water.

Total RNA purity and integrity assays

1 μ l of each of 2 RNA samples was subjected to 1% agarose gel electrophoresis for 80 V.times.20 min, and bands of 5s rRNA, 18s rRNA and 28s rRNA of total RNA were observed by gel imaging, and if three bands were intact, it was confirmed that the total RNA extraction was intact. 1 mul of RNA sample is diluted by 50 times, and then OD value is measured on Shimadzu (Japan) UV-1750 ultraviolet spectrophotometer, and the ratio of OD260/OD280 is 1.8-2.0, so that the prepared RNA is relatively pure and has no protein pollution.

Reverse transcription reaction

Reverse transcription was performed using 4. mu.L of RNA template according to the following reaction system:

reaction conditions are as follows: the reaction was carried out at 37 ℃ for 15min and then at 85 ℃ for 5 s.

Fluorescent quantitative PCR reaction

Reaction conditions are as follows: pre-denaturation at 95 deg.c for 3min, annealing at 60 deg.c for 30s, and extension at 95 deg.c for 15s for 40 cycles.

FIG. 19 is a comparison of cell proliferation capacity of microvesicle cell complex cultures after rupture of the sac at various time points.

FIG. 20 is a graph showing the effect of osteoblast gene expression in microvesicle complexes.

Alkaline phosphatase (ALP) Activity and staining assays

Alkaline phosphatase (ALP) activity: washing with PBS for 3-4 times (3 min/time); 0.1% Triton X-100 for 20 minutes; transferring the solution to a new EP tube; centrifuging at 14000rpm/min at 4 deg.C for 20 min; the supernatant was discarded, the pellet was transferred to a new EP tube, an ALP reaction substrate (20. mu.L of p-nitrophenyl 5m M phosphate, 20. mu.L of 5mM Mg Cl2 and 100. mu.L of 1M Tris-HCl) was added, and incubation at 37 ℃ for 30min in an incubator; adding NaOH to terminate the reaction, and detecting the absorbance value at 410nm by using an enzyme-labeling instrument; the protein content in the corresponding samples was determined using the BCA method, and the activities of alkaline phosphatase in each group were compared by dividing the absorbance by the protein content.

Staining with alkaline phosphatase (ALP), washing with PBS for 1-2 times, fixing with cold propanol for 10min, washing with sterile distilled water for 3-4 times, adding into ALP culture solution, and incubating at 37 deg.C for 3-10 h. Washing with tap water. Incubate 2% cobalt nitrate at 37 ℃ for 5min and wash with distilled water for 4 min. Incubating in 1% ammonium sulfide for 2min, washing with tap water, naturally drying, and sealing.

Alizarin red staining, namely removing the culture medium by suction, and washing for 2 times by PBS; fixing 4% neutral formaldehyde at room temperature for 30 min; washing with distilled water for 2 times; adding 1% alizarin red solution, and dyeing for 20 min; discard the staining solution, wash with PBS 2-3 times, observe under optical microscope and take pictures.

Von Kossa staining, which is to discard the original culture medium and wash with PBS 3 times; fixing with 4% neutral formaldehyde for 30 min; rinsing with double distilled water for 2-3 times; adding 1mL of Von Kossa dye solution per hole; irradiating with ultraviolet for 40 min; washing with distilled water for 3-5 min; dropping hematoxylin staining solution for counterstaining for 5 min; washing with tap water for 1 min; returning the diluted ammonia water to blue for several seconds, and washing for 2 min; dyeing with eosin dye solution for 3-5 min; the photographs were observed under an optical microscope.

FIG. 21 is a graph showing the effect of alkaline phosphatase (ALP) activity.

FIG. 22 is an alkaline phosphatase (ALP) staining, wherein FIG. 22A shows that no significant alkaline phosphatase is observed after 7 days of osteogenic induction of normal BMSCs; FIG. 22B shows that after 14 days of osteogenic induction of BMSCs, alkaline phosphatase staining partially appears positive; FIG. 22C shows a small number of positive alkaline phosphatase cells after 7 days osteogenic induction of BMP 2/Tet-on-BMSCs; FIG. 22D shows that alkaline phosphatase concentrated staining of BMP2-BMSCs was more pronounced after 14 days of osteogenic induction than that of regular BMSCs.

Fig. 23 is alizarin red staining, wherein fig. 23A shows alizarin red staining after 14 days of induction of normal BMSCs in the microvesicles, with formation of a small amount of calcium nodules visible; FIG. 24C shows alizarin red staining after 14 days of induction of BMP2-BMSCs in the microcapsules, and obvious calcium nodules are found.

Promotion of mineral salt deposition after osteogenic induction (Von kossa staining) is shown in fig. 24, wherein in fig. 24, Von kossa staining was observed 14 days after induction of ordinary BMSCs in the microcapsules, and a small amount of mineral salt deposition was observed; FIG. 24C shows that the BMP2/Tet-on-BMSCs stain with Von kossa after 14 days of induction in the microcapsules, and mineral salt deposition is evident.

And (4) conclusion:

as shown in FIGS. 6-11, the best MOI value of hBMP-2-GV347 lentiviral vector transfection is 50, the best puromycin screening concentration is 3 μ g/ml, the proliferation capacity of transfected BMSC cells is stronger than that of ordinary BMSC, and BMP-2 protein and RNA are continuously and stably expressed under the induction of DOX.

The cell growth curve analysis shows that the growth proliferation capacity of the BMSCs in the experimental group treated by the lentivirus containing BMP-2 and Tet-On systems has no statistical significance with the BMSCs group which only contains GFP and acts by the lentivirus and the blank control group (P is more than 0.05). The observation of the fluorescence of the transfected cells under a fluorescence microscope shows that the lentivirus titer with the MOI of 50 can not only ensure that the transfection efficiency of the cells reaches more than 90 percent, but also ensure that the cell proliferation is not obviously influenced. After BMSCs are transfected by lentivirus containing BMP-2 gene, BMP-2 gene expression and protein secretion are obviously increased (P <0.05), and alkaline phosphatase (ALP) activity and calcium nodule and mineral salt deposition are obviously more than those of common BMSCs and GFP transfection groups. Westren blot detection also proves that the Tet-on/BMP-2-GV347 transfection group expresses more obviously than untransfected MSCs; in addition, BMSCs transfected by Tet-on/BMP-2-GV347 highly express osteogenesis related markers: runx2 and OCN.

TABLE 3 transfection efficiency of lentiviruses at different MOI values

BMP2 gene expression was examined by q-PCR in BMSCs at various time points after transfection. The q-PCR results showed that transfection of Lentiviral-BMP2 significantly increased BMP-2 gene expression.

q-PCR showed significant increase in BMP2 gene expression in the Lentiviral-BMP2 group (P <0.05) 7 and 14 days after Lentiviral transfection.

Western-blot shows that BMSCs transfected by BMP-2 gene express BMP-2 protein intracellularly, while untransfected BMSCs do not express protein.

BMSCs transfected with Tet-on/BMP-2-GV347 highly expressed osteogenesis-related markers: runx2 and OCN.

The diameter of the APA microcapsule complex prepared by a high-voltage electrostatic field chemical method is (562 +/-69) mu m, and the diameter of the CMC-Ph microcapsule complex prepared by a microfluid method is (106 +/-15) mu m; comparing the two groups of microcapsules by a high-speed centrifugation method to obtain the complete rate of the composite of the two groups of microcapsules, the CMC-Ph microcapsule complex has stronger mechanical strength. The survival rate of cells in the APA microcapsule complex is in negative linear correlation with time, and is close to 0 at 28 days, the survival rate of BMSCs cells in the CMC-Ph microcapsule complex in 28 days is more than 50%, and survival cells can be observed till 54 days. The phenol-containing carboxymethyl cellulose (CMC-Ph) is successfully prepared into monodisperse microcapsules by coaxial copolymerization microfluid under the control of a micro-fluid pump; when the flow rate of the liquid paraffin is controlled to be 12ml/min and the flow rate of the CMC-Ph mixture is controlled to be 60 mu L/min, microcapsules with the diameter of 80-160 mu m can be prepared, and the microcapsules are circular and uniform in size; the survival rate of the cells in the microcapsule is (87.41 +/-2.52)%, and the survival rate after two weeks is (84.76 +/-4.83)%. The cells in the microcapsule are proliferated and clustered at 2 weeks under a microscope; most of the cells in the microcapsules survive when observed under a fluorescence microscope for 21 days (green fluorescence); the mitochondria of the cells in the microcapsule are actively metabolized, and high expression still exists after 3 weeks. The secretion of BMP-2 protein is continuously increased along with the prolonging of time under the action of DOX, the peak is reached in 14 to 15 days, and then the protein gradually falls back; when DOX is stopped, the secretion of BMP-2 protein by the cells in the microcapsules is rapidly stopped; there was no significant difference in the amount of BMP-2 protein in the medium secreted by the microencapsulation and the individual cells (P > 0.05).

The growth and proliferation capacities of the cells after the capsulorhexis at each time point are different but the difference is not significant (P is more than 0.05); after osteogenic induction, high expression of ALP gene, type I collagen (COLL I), bone calcium (OC) gene, bone bridge (OPN) gene and RUNX2 gene are generated; alkaline phosphatase staining, calcium nodule and calcium salt deposit staining all indicated that microencapsulated BMP2-BMSCs were more easily transformed into osteoblasts than untransfected cells.

SEQUENCE LISTING

<110> Guangzhou medical university affiliated first hospital

<120> method for constructing microencapsulated BMP-2/Tet-on system

<130>

<160> 27

<170> PatentIn version 3.3

<210> 1

<211> 46

<212> DNA

<213> BMP2(9288-1)-P1

<400> 1

aaccgtcaga tcgcaccggc gccaccatgg tggccgggac ccgctg 46

<210> 2

<211> 40

<212> DNA

<213> BMP2(9288-1)-P2

<400> 2

tcaccatggt ggcgaccggg cgacacccac aaccctccac 40

<210> 3

<211> 18

<212> DNA

<213> KL9288-P3

<400> 3

ctgtgatgcg gtggactg 18

<210> 4

<211> 22

<212> DNA

<213> pEGFP-N-3

<400> 4

cgtcgccgtc cagctcgacc ag 22

<210> 5

<211> 22

<212> DNA

<213> H-BMP-2-Forward Primer

<400> 5

aaaacgtcaa gccaaacaca aa 22

<210> 6

<211> 23

<212> DNA

<213> H-BMP-2-Reverse Primer

<400> 6

tccacgtaca aagggtgtct ctt 23

<210> 7

<211> 21

<212> DNA

<213> H-βactin-Forward primer

<400> 7

gcatgggtca gaaggattcc t 21

<210> 8

<211> 20

<212> DNA

<213> H-βactin-Reverse Primer

<400> 8

tcgtcccagt tggtgacgat 20

<210> 9

<211> 20

<212> DNA

<213> R-OC-Forward Primer

<400> 9

ctcactctgc tggccctgac 20

<210> 10

<211> 20

<212> DNA

<213> R-OC-Forward Primer

<400> 10

ctcactctgc tggccctgac 20

<210> 11

<211> 20

<212> DNA

<213> R-OC-Reverse Primer

<400> 11

ccctcctgct tggacatgaa 20

<210> 12

<211> 22

<212> DNA

<213> R-RUNX2-Forward primer

<400> 12

aggcacagac agaagcttga tg 22

<210> 13

<211> 20

<212> DNA

<213> R-RUNX2-Reverse primer

<400> 13

aggcgggaca cctactctca 20

<210> 14

<211> 20

<212> DNA

<213> R-GAPDH-Forward Primer

<400> 14

agggctgcct tctcttgtga 20

<210> 15

<211> 21

<212> DNA

<213> R-GAPDH-Reverse Primer

<400> 15

aacttgccgt gggtagagtc a 21

<210> 16

<211> 20

<212> DNA

<213> R-ALP-Forward primer

<400> 16

gcacgacaat cgggatgaac 20

<210> 17

<211> 19

<212> DNA

<213> R-ALP-Reverse primer

<400> 17

tccagcagga cggtcatca 19

<210> 18

<211> 20

<212> DNA

<213> R-OC-Forward Primer

<400> 18

ctcactctgc tggccctgac 20

<210> 19

<211> 20

<212> DNA

<213> R-OC-Reverse Primer

<400> 19

ccctcctgct tggacatgaa 20

<210> 20

<211> 20

<212> DNA

<213> R-Collagen I-Forward Primer

<400> 20

ctcccagcgg tggttatgac 20

<210> 21

<211> 19

<212> DNA

<213> R-Collagen I-Reverse Primer

<400> 21

tgctggctca ggctcttga 19

<210> 22

<211> 20

<212> DNA

<213> R-OPN-Forward primer

<400> 22

gctgacgctg gaaagttgga 20

<210> 23

<211> 20

<212> DNA

<213> R-OPN-Reverse primer

<400> 23

ttcctcgtcg ctttccttca 20

<210> 24

<211> 22

<212> DNA

<213> R-RUNX2-Forward primer

<400> 24

aggcacagac agaagcttga tg 22

<210> 25

<211> 20

<212> DNA

<213> R-RUNX2-Reverse primer

<400> 25

aggcgggaca cctactctca 20

<210> 26

<211> 20

<212> DNA

<213> R-GAPDH-Forward Primer

<400> 26

agggctgcct tctcttgtga 20

<210> 27

<211> 21

<212> DNA

<213> R-GAPDH-Reverse Primer

<400> 27

aacttgccgt gggtagagtc a 21

Claims (1)

1. A method for constructing a microencapsulated hBMP-2/Tet-on system is characterized by comprising the following steps:

s1, designing a target gene hBMP-2 primer, directionally cloning the target gene hBMP-2 to a GV347 vector carrying a Tet-on regulation system after PCR amplification and purification, and constructing a hBMP-2-GV347 plasmid;

s2, transforming and amplifying the hBMP-2-GV347 plasmid, and co-infecting 293T cells with the helper plasmid; collecting and concentrating lentivirus liquid, and transfecting 293T cells by using a lentivirus vector to obtain a lentivirus vector containing hBMP-2-GV 347;

s3, transfecting BMSCs with the lentivirus vector containing hBMP-2-GV347 to obtain hBMP-2-GV 347-BMSCs;

s4, encapsulating hBMP-2-GV347-BMSCs by using carboxymethyl cellulose containing phenolic groups as a microcapsule material;

wherein the particle size of the phenolic carboxymethyl cellulose microcapsule in S4 is 80-160 μm;

the preparation method of the phenolic carboxymethyl cellulose microcapsule comprises the following steps: using a liquid copolymerization microfluid device and a micro-injection pump as generators, using liquid paraffin as external fluid, using a carboxymethyl cellulose solution containing phenol groups as internal fluid, standing the phenol-based carboxymethyl cellulose microcapsules for 20-30min, discarding the liquid paraffin after the microcapsules are completely deposited at the bottom of a collecting pipe, adding a PBS solution, and performing centrifugal washing on a centrifugal machine at 2000rpm/min multiplied by 5 min;

the microcapsule package in S4 specifically comprises: selecting hBMP-2-GV347-BMSCs 1 x 10⁷The cell suspension is digested for standby; dispersing phase is 0.1g of phenolic carboxymethyl cellulose, dissolving in 4ml of L-DMEM, and magnetically stirring for 30min until completely dissolving; adding 1mg/mL horseradish peroxidase, and fully dissolving; adjusting the flow rate on the micro pump: the dosage of the continuous phase is 16 mL/min; the dosage of the dispersed phase is 50 mu L/min; repeatedly centrifuging, standing and washing.

Images