Uncle Hu
Editor|Uncle Hu
preface
Bone defects are a common disease, and its effective repair has always been a problem in the field of orthopedics. At present, the most common treatment of bone defects is autologous and allogeneic bone.
However, at present, the problems of single source of autologous bone, donor bone defect and difficult to form are commonly used in clinical practice, and allogeneic bone marrow mesenchymal stem cells have the characteristics of easy infection and rejection, and neither of them can well adapt to clinical needs.
At present, traditional organic/inorganic composite scaffolds are difficult to take into account the characteristics of microporous structure, biocompatibility, degradability, controllability and mechanical strength.
How to develop a new type of bionic scaffold that can simulate the structure of human bones has become a hot issue in bone tissue engineering.
Growth factor (BMP-2) is the BMPs in the family of BMPs currently found to contribute to bone activity. The U.S. FDA has approved rhBMP-2 (rhBMP-2) for clinical use.
rhBMP-2 is a low molecular weight that is easily broken down quickly by water-soluble proteases in vivo, resulting in a short half-life of BMP-2 in the body and inability to maintain bone inducibility.
In addition, transplanting it into the human body in the form of foreign proteins, in the case of a large number of uses, there are certain toxic side effects and immune rejection and other problems.
At present, scholars at home and abroad have developed different forms of rhBMP-2 sustained release systems, but all have different degrees of defects. For example, the copolymers of polylactic acid and polyglycolic acid, etc., they have bone conduction ability and degradability.
When they are used as vectors, most rhBMP-2s are dispersed into tissues early in the cultivation process, so they are fast during the initial decomposition process.
In addition, the polymer degradants synthesized by them are acidic, which will cause a decrease in local pH, resulting in aseptic inflammation and foreign body reactions of local tissues, which has a certain impact on new bone formation.
Osteoblasts transconverted to the BMP-2 gene can maintain high expression of BMP-2 for a period of time, thus compensating for the above shortcomings.
Our research group combined BMSCs in BMP-2 genetically engineered dogs with calcium phosphate bone cement in the early stage and transplanted them into large-scale bone defects, which can effectively promote the healing of large-scale bone defects.
Therefore, we intend to construct a recombinant lentivirus of BMP-2 and introduce it into rat BMSCs to achieve sustained and stable expression of BMP-2.
At the same time, the GFP gene is introduced into the expression system of the gene, so that the GFP gene expressed can be expressed in vitro, so as to realize the real-time tracking, screening and enrichment of GFP gene expression.
Experimental materials
There were 10 SPF type non-specific pathogenic rats produced by Zhaoyan Pharmaceutical (Suzhou) Pharmaceutical Company, weighing 250-280g, 6-9 weeks old.
In accordance with the relevant provisions of the Ministry of Science and Technology of the People's Republic of China "Guiding Opinions on the Good Treatment of Laboratory Animals", corresponding treatment was carried out.
Rats with a body weight of 250g~280g aged 6~9 weeks were taken and BMSCs of cultured rats were isolated by Percoll cell isolation solution, density gradient centrifugation and differential adherent sub-screening.
At a rate of 1500 rpm, centrifuge for 15 minutes. Suspend 3 mL of medium again and slowly add to a centrifuge containing the same amount of Percoll cell isolate, followed by 30 min centrifugation at 2,000 r/min.
Interfacial layer cells are collected, rinsed twice with DMEM medium, and after centrifugation, the supernatant is discarded, and mixed with DMEM medium containing fetal bovine serum.
At a ratio of 4×105 cells/cm2, they were seeded in cell culture flasks and cultured in a cell culture incubator with a CO2 volume concentration of 5%, a temperature of 37°C, and saturated humidity. After the adhesion rate of the medium is 90%, it is enzymatically hydrolyzed by pancreatic enzymes and then passaged.
pHBLV-U6-GFP-PGK-Puro was selected as the cloning vector for plasmids. In this study, XhoI/BamHI was the research object, and the BMP-2 gene was used as the entry point.
Shanghai GK Biomedical Technology Co., Ltd. completed the plasmid construction, sequence analysis, packaging, concentration and viral titer detection of the target gene.
On the first day, the cells are digested with pancreatic protease and cultured in DMEM at concentrations from 6x106 cells/ml to 8x106 cells/ml.
Well-preserved 3 generations of bone marrow mesenchymal stem cells (BMSCs) were seeded on 6-well plates with 5x105 cells inside each well and cultured overnight at 37 °C.
On the second day, when the cells were glued together, they were dissolved again, and the viral titer was detected to be 5x108TU/mL and the molar index was 20, and then 20 μl of viral liquid was slowly added to the cells and mixed evenly. Place it in the incubator and start further incubation.
After reverse transcription, it is amplified with BMP-2 primers. In one sterile PCR tube, add 2 μL sequentially, 2 μL of upstream primers, 2 μL of downstream primers, 5 μL of PCR buffer, 1 μL of Taq polymerase, and replenish enough deionized water to achieve a total capacity of 50 μL.
Based on the previous work, this project intends to use rat bone marrow mesenchymal stem cells (BMSCs) as the research object, and actinβ-actin as the internal reference.
Intracellular expression of BMP-2 seeds stable, transfected and untransfected BMSCs in a cell culture flask at a concentration of 1×105 cells/cm2.
When the cells grow to 90% confluency, they are digested with pancreatic enzymes, and cytosols are added to the collected cells, and the solute is centrifuged at a rate of 12000r/min for 15min at 4 °C, and the supernatant obtained is protein extract.
BMSCs were used as the research object, and the double antibody sandwich test was used to observe their effect on the target gene.
Rabbit anti-human BMP-2 antibody was coated on 96-well plates and stained with HRP and GAPDH-labeled sheep anti-rabbit IgG and DAB, respectively, and the absorption rate at 460 nm was determined by enzyme labeling.
The role of unexpressed and non-expressed BMSCs in in vitro induced differentiation was observed. These cells are seeded on five 96-well dishes at a concentration of 2x103 pcs per ml.
The MTT method was used to observe the growth of in vitro cultured BMSCs.
Dimethyl sulfoxide is added to each well to dissolve it into crystals. The vibrator vibrates for 10 minutes, shakes evenly, and reads out and absorbs by enzyme labeling at each pore size at 490 nm.
The third generation is a long spindle-shaped fibroblast that grows in a spiral and radial manner and multiplies rapidly after full fusion, as shown in Figure 1.
As shown in Figure 2, the expression of BMP-2 is without any green light before transfection, and after transfection, the expression of BMP-2 takes on a green light under the fluorescence microscope of BMSCs, demonstrating that the transfection of BMSCs is successful.
After induced transfection, the histological changes were consistent with expectations.
The expression of BMP-2mRNA is shown in Figure 3. RT-PCR detection showed that the expression of genetically modified bone marrow matrix metalloproteinase-2 gene was significantly increased compared with that of wild type.
Compared with the control group, the expression level of BMP-2 mRNA was significantly higher than that of the control group (P<0.05).
The western blot results are shown in Figure 4, and rabbit anti-human BMP-2 antibody and GAPDH-labeled sheep anti-rabbit IgG antibody were hybridized with rabbit anti-human BMP-2 antibody, respectively.
The results showed that the prepared recombinant plasmid could form bands in vitro, which matched the desired results. In contrast, only GAPDH bands were detected in the non-transfected group, while BMP-2 was not expressed.
The absorbance of each well was read in with a microplate reader and the mass concentration of the BMP-2 protein of interest in the cell culture supernatant was calculated based on a standard curve (see Figure 5).
As can be seen from Figure 6, the mass concentration of BMP-2 in the culture supernatant of transfected cells is significantly higher than in the culture supernatant of transfected cells (P<0.05).
Our previous research found that succinate dehydrogenase converts MTT into an insoluble blue amethyst crystal form in living mitochondria and precipitates in living mitochondria, while dead cells do not.
Sodium dimethylsulfinate can dissolve the methyl isomer in bacteria, and the enzyme labeling method can directly reflect the absorption of bacteria at 490nm, which can be used to determine the number of bacteria.
Under a certain number of somas, the formation of MTT crystals has a linear relationship with the number of somas. As can be seen from Figure 7, the proliferation rate of BMSCs in the transfected group is significantly higher than that of BMSCs and empty carrier BMSCs in the untransfected group (P<0.05).
Although BMSCs come from a wide range of sources, their proportion in vivo is small, and in order to meet clinical requirements, researchers often use in vitro isolation, purification and amplification methods to improve the quality of BMSCs.
At present, the internationally widely used separation technologies include adherent method, density gradient centrifugation method and surface molecular marker method.
In this experiment, BMSCs were separated by density gradient centrifugation, and then the cells were purified by adherent screening and separation, which was easy to operate and obtained a large number of BMSCs with stable performance. Compared with conventional methods, the use of genetic methods for restoration has obvious advantages.
BMP-2 is one of the most widely studied and in-depth osteogenic factors. Our previous study found that the BMP-2 gene has a high expression in BMSCs. After the detection of BMP-2 expression by MTT, the growth rate of BMSCs was significantly increased (P<0.0.05).
Bibliography:
[1] HUANG Junfeng, WANG Daping, HE Chunyu, et al. "In vitro construction of adenovirus-mediated bone morphogenetic protein 2 gene transfection bone marrow mesenchymal stem cells composite nano-hydroxyapatite bone grafting materials"
[2] Zhang Gang, Lu Laichun, Qiu Songbo, et al., "Preparation and Biological Effects of rhBMP-2-PLA Nanospheres"
[3] Qiu Lin, Jin Xianqing, "Biological characteristics of bone marrow mesenchymal stem cells and their clinical therapeutic application"