Objective To review researches of BMSCs in tumor therapy. Methods The recent relevant l iterature was extensively reviewed. The tropism of BMSCs to cancer, the effect of BMSCs on tumor growth and the appl ication of BMSCs in tumor therapy were summarized. Results BMSCs has the tropism to tumor and may inhibit or enhance growth of tumor. BMSCs as gene-del ivery vehicle for gene therapy had obtained certain therapeutic efficacy. However, BMSCs can become tumorigenic. Conclusion BMSCs is a good gene-del ivery vehicle for gene therapy. The relationship of BMSCs and tumorcells should be studied deeply for enhance the safety of BMSCs in gene therapy of tumor.
To study the effect of the repair of rabbit articular cartilage defects by the composite of chondrogenic induction of autologous MSCs and autologous “two-phase” bone matrix gelatin (BMG). Methods Twentyfour healthy adult New Zealand rabbits weighing 2 to 3 kg were divided into group A, B and C with 8 in each. Autologous MSCsderived from group A were cultured in vitro and observed under inverted phase contrast microscope when enough cells through trypsinization transferring in vitro were obtained. Then the growth curves of 1, 3 and 5 passage culture of MSCs were drawn. The 3rd passage MSCs were induced into chondrogenic differentiation by adding TGF-β1 (10 ng/mL), IGF-1 (10 ng/mL) and vitamin C (50 ng/mL) in vitro. At 8 days after induction, the features of chondrocytes were observed under inverted phase contrast microscope, and immunohistochemical staining and Mallory staining were made. Getting out part of the il ium of group A and B, according to the method of Urist, the “two-phase” BMG was acquired. Chondrogenic induction of autologous MSCs was inoculated into the corresponding BMG to set up a composite of cell-carrier, and then it was observed through scanning electric microscope after 3 days of culture. The model of articular cartilage defects of rabbits was made: in group A, autologous cell-carriers were implanted; in group B, there only existed autologous BMG; in group C, there was nothing. At 8, 12 weeks after operation, the gross, HE staining and immunohistochemical staining were made, and grading scales were evaluated according to Wakitani histological grading method. Results Features of MSCs were as follows: the shape of primary cells was shotspindled and of passage cells was long. As to the growth curves of 1, 3 and 5 passage culture of MSCs, passage cells grew slowly for 3 days after being passaged and went into log-growth during the 3rd and the 7th days and into plateau later, but the 3rd passage cells grew best. Observation of MSCs after chondrogenic induction was performed: the shape of cells was ell iptical and the effect of induction was verified by the positive results of collagen type II, S-100 and Mallory staining. Under scanning electricmicroscope, the structure of BMG was good and cells were observed growing in it well. As far as repair of articular cartilage defects are concerned at 8, 12 weeks after transplantation, the defects in group A were repaired by the hyl ine-l ike tissue and the structures of the cartilage surface and normal cartilage were in integrity, and immunohistochemical staining of collagen type II was positive, while those in group B and C were repaired by the fibrous-l ike tissues and the surfaces were irregular. In Wakitanni histological score, at 8 weeks after operation, group A was (3.50 ± 1.51) points, group B was (10.00 ± 1.41) points and group C was (12.00 ± 0.93) points; at 12 weeks, group A was (1.13 ± 0.99) points, group B was (8.38±1.30) points, and group C was (10.13 ± 1.64) points. At different time points, group A was significantly better than group B and C, showing significant differences (P lt; 0.05). Conclusion Induced autologous MSCs and the composite with autologous “two-phase” BMG have the function to repair articular cartilage defects, and they are better than autologous BMG transplanted only or nothing transplanted.
Objective To introduce growth and differentiation factor 5 (GDF-5) gene into hBMSCs using recombinant adenovirus vector and to investigate the effect of GDF-5 gene expression on hBMSCs osteogenic differentiation. Methods Recombinant adenovirus GDF-5 (Ad-GDF-5) containing green fluorescent protein (GFP) and Ad-GFP were amplifiedand tittered. hBMSCs at passage 3 were infected with two viruses at different titers. At 2 days after intervention, GFP expression was observed using fluorescence microscope, and GDF-5 expression in hBMSCs was detected by RT-PCR. Adherent hBMSCs at passage 3 were randomly divided into 4 groups: experimental group (GDF-5 gene transfection), osteogenic induction group, Ad- GFP infection group, and control group. Cell differentiation was detected by inverted phase contrast microscope observation, fluorescence microscope observation, reverse transcription fluorescence quantitative PCR, immunofluorescence staining, and von Kossa staining at different time points after intervention. Results The titer of Ad-GDF-5 and Ad-GFP was 1.0 × 109 pfu/mL and 1.2 × 109 pfu/mL, respectively. hBMSCs was efficiently infected by Ad-GDF-5 and Ad-GFP, and expressed target gene and GFP gene. At 1-7 days after intervention, morphology and growth pattern of the hBMSCs in the experimental group and the osteogenic induction group were transformed into osteoblast-l ike cells, whereas the cells in the other two groups were still maintained their original morphology and growth pattern. Reverse transcription fluorescence quantitative PCR detection: at 4 days after intervention, GDF-5 expression in the experimental group was obviously higher than that of other groups (P lt; 0.05); ALP, Col I, and OC gene expression in the experimental and the osteogenic induction group were superior to those of theAd-GFP infection and the control group (P lt; 0.05); Col I gene expression in the osteogenic induction group was greater than that of the experimental group (P lt; 0.05). Immunofluorescence staining: at 4 days after intervention, the cells in the osteogenic induction group and the experimental group expressed and secreted Col I, and no expression of Col I was evident in the other two groups. At 10 days after intervention, the cells in the osteogenic induction and the experimental group were positive for von Kossa staining, and the results of the other two groups were negative. Conclusion GDF-5 gene can be transferred into hBMSCs via adenovirus vector and be expressed stably. It can facil itate the osteogenic differentiation of the hBMSCs and lay a foundation for the further study of this kind of gene transferred hBMSCs effect on bone tissue repair.
【Abstract】 Objective To investigate the secretion of target gene and differentiation of BMSCs transfected by TGF-β1 and IGF-1 gene alone and together into chondrocytes and to provide a new method for culturing seed cells in cartilage tissue engineering. Methods The plasmids pcDNA3.1-IGF-1 and pcDNA3.1-TGF-β1 were ampl ified and extracted, then cut by enzymes, electrophoresed and analyzed its sequence. BMSCs of Wistar rats were separated and purificated by the density gradient centrifugation and adherent separation. The morphologic changes of primary and passaged cells were observed by inverted phase contrast microscope and cell surface markers were detected by immunofluorescence method. According to the transfect situation, the BMSCs were divided into 5 groups, the non-transfected group (Group A), the group transfected by empty vector (Group B), the group transfected by TGF-β1 (Group C), the group transfected by IGF-1 (Group D) and the group transfected both by TGF-β1 and IGF-1 (Group E). After being transfected, the cells were selected, then the prol iferation activity was tested by MTT and expression levels were tested by RT-PCR and Western blot. Results The result of electrophoresis showedthat sequence of two bands of the target genes, IGF-1 and TGF-β1, was identical with the sequence of GeneBank cDNA. A few adherent cells appeared after 24 hours culture, typical cluster formed on the forth or fifth days, and 80%-90% of the cells fused with each other on the ninth or tenth days. The morphology of the cells became similar after passaging. The immunofluorescence method showed that BMSCs were positive for CD29 and CD44, but negative for CD34 and CD45. A few cells died after 24 hoursof transfection, cell clone formed at 3 weeks after selection, and the cells could be passaged at the forth week, most cells became polygonal. The boundary of some cells was obscure. The cells were round and their nucleus were asymmetry with the particles which were around the nucleus obviously. The absorbency values of the cells tested by MTT at the wavelength of 490 nm were0.432 ± 0.038 in group A, 0.428 ± 0.041 in group B, 0.664 ± 0.086 in group C, 0.655 ± 0.045 in group D and 0.833 ± 0.103 in group E. The differences between groups A, B and groups C, D, E were significant (P lt; 0.01). The differences between groups A and B or between C, D and E were not significant (P gt; 0.05)。RT-PCR and Western blot was served to detect the expression of the target gene and protein. TGF-β1 was the highest in group C, 0.925 0 ± 0.022 0, 124.341 7 ± 2.982 0, followed by group E, 0.771 7 ± 0.012 0, 101.766 7 ± 1.241 0（P lt; 0.01）; The expression of IGF-1 was the highest in group E, 1.020 0 ± 0.026 0, 128.171 7 ± 9.152 0, followed by group D, 0.465 0 ± 0.042 0, 111.045 0 ± 6.248 0 （P lt; 0.01）. And the expression of collagen II was the hignest in group E, 0.980 0 ± 0.034 0, 120.355 0 ± 12.550 0, followed by group C, 0.720 0 ± 0.026 0, 72.246 7 ± 7.364 0（P lt; 0.01）. Conclusion The repairment of cartilage defects by BMSCs transfected with TGF-β1 and IGF-1 gene together hasa good prospect and important significance of cl inic appl ication in cartilage tissue engineering.
Objective To compare the molecular phenotype of human intervertebral disc cells and articular chondrocytes and to analyze whether hBMSCs can differentiate into both chondrocytes and nucleus pulposus cells after combined induction of TGF-β3 and BMP-7 in vitro. Methods The cells with the characteristics of hBMSCs were isolated from marrow aspirates of the volunteer donors’ il iac crest. Human bone marrow was removed and fractionated, and adherent cell cultures were establ ished. The 4th passage cells were then translated into an aggregate culture system in a serum-free medium. The pellet cultures of hBMSCs were divided into four groups: 10 ng/mL TGF-β3 group (group A), 200 ng/mL BMP-7 group (group B), combination group of TGF-β3 and BMP-7 (group C) and blank group as the control (group D). Histological observation, RT-PCR and RQ-PCR were appl ied to measure the expressions of collagen type I, II, X, aggrecan and SOX9 on the 4th and 21st day after cell induction, respectively. Results As was shown by histological observation, the induced cells expressed the feature of chondrocytes in morphology and ECM in groups A and C on the 21st day after the culture. And the collagen type II was positive after staining in groups A and C. The cell morphology of the induced cells in groups B and C had no obviouly changed. PCR detection showed that the expressions of SOX9, aggrecan, collagen type I, II in groups A and C at 21st day were more increased than those at 4th day (P lt; 0.05). The only expressions of collagen type I in groups B and D at 21st day were more increased than those at 4th day (P lt; 0.05). The expressions of collagen type X only was positive in group A. Conclusion Combination of TGF-β3 and BMP-7 can make the differentiated cells from hBMSCs much closer to intervertebral disc cells, so it perhaps could provide seed cells for intervertebral disc tissue engineering.
Objective To develop three-dimensional (3D) porous nanofiber scaffold of PLGA-silk fibroincollagen and to investigate its cytocompatibil ity in vitro. Methods Method of electrostatic spinning was used to prepare 3D porous nanofiber scaffold of PLGA-silk fibroin-collagen (the experimental group) and 3D porous nanofiber scaffold of PLGA (the control group). The scaffold in each group was observed by scanning electron microscope (SEM). The parameters of scaffold fiber diameter, porosity, water absorption rate, and tensile strength were detected. SC harvested from the bilateral brachial plexus and sciatic nerve of 8 SD suckl ing rats of inbred strains were cultured. SC purity was detected by S-100 immunohistochemistry staining. The SCs at passage 4 (5 × 104 cells/mL) were treated with the scaffold extract of each group at a concentration of 25%, 50%, and 100%, respectively; the cells treated with DMEM served as blank control group. MTT method was used to detect absorbance (A) value 1, 3, 5, and 7 days after culture. The SC at passage 4 were seeded on the scaffold of the experimental and the control group, respectively. SEM observation was conducted 2, 4, and 6 days after co-culture, and laser scanning confocal microscope (LSCM) observation was performed 4 days after co-culture for the growth condition of SC on the scaffold. Results SEM observation: the scaffold in two groups had interconnected porous network structure; the fiber diameter in the experimental and the control group was (141 ± 9) nm and (205 ± 11) nm, respectively; the pores in the scaffold were interconnected; the porosity was 87.4% ± 1.1% and 85.3% ± 1.3%, respectively; the water absorption rate was 2 647% ± 172% and 2 593% ± 161%, respectively; the tensile strength was (0.32 ± 0.03) MPa and (0.28 ± 0.04) MPa, respectively. S-100 immunohistochemistry staining showed that the SC purity was 96.5% ± 1.3%. MTT detection: SC grew well in the different concentration groups and the control group, the absorbance (A) value increased over time, significant differences were noted among different time points in the same group (P lt; 0.05), and there was no significant difference between the different concentration groups and the blank control group at different time points (P gt; 0.05). SEM observation: in the experimental group, SC grew well on the scaffold, axon connection occurred 4 days after co-culture, the cells prol iferated massively and secreted matrix 6 days after co-culture, and the growth condition of the cells was better than the control group. The condition observed by LSCM 4 days after co-culture was the same as that of SEM. Conclusion The 3D porous nanofiber scaffoldof PLGA-silk fibroin-collagen prepared by the method of electrostatic spinning is safe, free of toxicity, and suitable for SC growth, and has good cytocompatibil ity and proper aperture and porosity. It is a potential scaffold carrier for tissue engineered nerve.
Objective To investigate the effect and mechanism of leptin (LEP) on the osteoblastic differentiation of hBMSCs in vitro. Methods Whole bone marrow culture method was appl ied to culture hMBSCs and hBMSCs at passage 3 were divided into groups A, B, C, D, E and F, and when cell attachment was evident, 400, 200, 100 and 50 ng/mL LEP, 100 ng/mLBMP and common nutrient medium were added into each group, respectively. ALP staining and mineral ized nodules staining were conducted at 7 and 21 days after culture, respectively. And inverted phase contrast microscope observation was performed. ALP activity and osteocalcin (OCN) level of hBMSCs in each group was detected at 7, 14 and 21 days after culture to select the best induced concentration of LEP on osteoblastic differentiation. For groups of B, E and F at 7 days after culture, RT-PCR was adopted to detect the expression of such osteogenesis-related genes as core-binding factor α 1 (Cbfα1), ALP, Col I and OCN mRNA. Results At 7 days after induced culture, the ALP staining result showed that the endochylema in groups A, B, C, D and E were stained blue and the endochylema in the group F was sl ightly positive. At 21 days after induced culture, the mineralized nodules staining showed that cells in groups A, B, C, D and E were stained positively and cells in group F were negative. At 7, 14 and 21 days after culture, ALP and OCN activities in group B were less than that of group E (P lt; 0.05), but significant higher than that of groups A, C, D and F (P lt; 0.05), the optimal concentration of LEP was 200 ng/mL. At 7 days after culture, group F witnessed no expression of Cbfα1, ALP, Col I and OCN mRNA, while groups B and E witnessed expressions of all those indexes, but the expressions in group B were less than those of group E. Conclusion LEP can stimulate osteoblastic differentiation of hBMSCs in vitro, and the possible mechanism is its role of promoting the expression of osteoblastic related genes.
Objective To investigate the effects of intermittent negative pressure on the mRNA expression of osteoprotegerin (OPG) and osteoprotegerin l igand (OPGL) in human BMSCs cultured in vitro. Methods BMSCs were isolated from adult marrow donated by 2 hip osteoarthritis patients with prosthetic replacement in January 2008 and cultured in vitro. The third passage cells were divided into experimental group and control group. The experimental group was induced by negative pressure intermittently for 2 weeks (pressure: 50 kPa, 30 minutes each time, twice per day) and the control groupwas routinely cultured. After 2 weeks of culture, cell morphology was observed by inverted phase contrast microscope, and the mRNA expressions of OPG and OPGL in BMSCs were analyzed by real-time PCR. Results The cell prol iferation speed of the experimental group was slower than that of the control group. The cell morph changed from shuttle to megagon with some prominences in experimental group and the cell morph kept shuttle in the control. The mRNA expression of OPG in experimental group increased significantly (P lt; 0.01) and the mRNA expression of OPGL in experimental group decreased significantly compared with control group (P lt; 0.01) 2 weeks later. Conclusion Intermittent negative pressure is capable of promoting the expression of OPG, while inhibiting the expression of OPGL in human BMSCs.
ObjectiveBenign familial epilepsies that present themselves in the first year of life include benign familial neonatal epilepsy (BFNE), benign familial neonatal-infantile epilepsy (BFNIE) and benign familial infantile epilepsy (BFIE). We aim to investigate gene mutations and the relationship between genotypes and clinical phenotypes in benign familial epilepsies in the first year of life.MethodsWe recruited families with benign familial epilepsies in the first year of life at Peking University First Hospital from September 2006 to January 2018. Clinical information and blood samples were obtained from probands and their family members. For BFIE families, mutation screening of PRRT2 was performed by using the polymerase chain reaction (PCR) and Sanger sequencing at first. The PRRT2 mutation negative probands of BFIE families were further screened for pathogenic mutations by targeted next-generation sequencing. The probands of BFNE and BFNIE families were screened for pathogenic mutations by targeted next-generation sequencing.ResultsA total of 89 families with benign familial epilepsies in the first year of life were collected. Of the 89 families, 4 were classified as BFNE, 7 as BFNIE, and 78 as BFIE. Genetic testing led to the identification of gene mutations in 68 families (76.4%), including 50 families had PRRT2 mutations (hotspot mutation c.649dupC was detected in 32 families; c.649delC was detected in 6 families), 9 families had KCNQ2 mutations, 8 families had SCN2A mutations, and one family had GABRA6 mutation. In the 4 BFNE families, causative mutations were only found in KCNQ2, which was identified as the causative gene in 3 families. The remaining one BFNE family was not detected with any pathogenic mutation. All 7 BFNIE families had identifiable gene mutations, KCNQ2 was found in 3 families, SCN2A in 3 families, and PRRT2 in one family. In the 78 BFIE families, gene mutations were identified in 58 families (74.4%), with PRRT2 mutations found in 49 families (62.8%), SCN2A mutations found in 5 families, KCNQ2 mutations found in 3 families, and a novol GABRA6 mutation found in one family. Twenty BFIE families were not identified with any gene mutations. In 78 BFIE families, 18 were subclassified as infantile convulsions with paroxysmal choreoathetosis syndrome(ICCA). 17 of 18 ICCA families were detected with PRRT2 mutations (17/18, 94.4%). The remaining ICCA family was not detected with any pathogenic mutation.ConclusionsOur results confirmed that mutations in KCNQ2, SCN2A, and PRRT2 are major genetic causes of benign familial epilepsy in the first year of life in the Chinese population. KCNQ2 is the major gene related to BFNE. PRRT2 is the main gene responsible for BFIE. KCNQ2 and SCN2A mutations are common in BFNIE families. GABRA6 mutation might be a new cause of BFIE. Identification of underlying gene mutation can be helpful for clinical diagnosis and judgement of the prognosis.
Objective To observe the effect of BMSCs on the cardiac function in diabetes mellitus (DM) rats through injecting BMSCs into the ventricular wall of the diabetic rats and investigate its mechanism. Methods BMSCs isolated from male SD rats (3-4 months old) were cultured in vitro, and the cells at passage 5 underwent DAPI label ing. Thirty clean grade SD inbred strain male rats weighing about 250 g were randomized into the normal control group (group A), the DM group (group B), and the cell transplantation group (group C). The rats in groups B and C received high fat forage for 4 weeks and the intraperitoneal injection of 30 mg/kg streptozotocin to made the experimental model of type II DM. PBS and DAPI-labeledpassage 5 BMSCs (1 × 105/μL, 160 μL) were injected into the ventricular wall of the rats in groups B and C, respectively. After feeding those rats with high fat forage for another 8 weeks, the apoptosis of myocardial cells was detected by TUNEL, the cardiac function was evaluated with multi-channel physiology recorder, the myocardium APPL1 protein expression was detected by Western blot and immunohistochemistry test, and the NO content was detected by nitrate reductase method. Group C underwent all those tests 16 weeks after taking basic forage. Results In group A, the apoptosis rate was 6.14% ± 0.02%, the AAPL1 level was 2.79 ± 0.32, left ventricular -dP/dt (LV-dP/dt) was (613.27 ± 125.36) mm Hg/s (1 mm Hg=0.133 kPa), the left ventricular end-diastol ic pressure (LVEDP) was (10.06 ± 3.24) mm Hg, and the NO content was (91.54 ± 6.15) nmol/mL. In group B, the apoptosis rate was 45.71% ± 0.04%, the AAPL1 level 1.08 ± 0.24 decreased significantly when compared with group A, the LVdP/ dt was (437.58 ± 117.58) mm Hg/s, the LVEDP was (17.89 ± 2.35) mm Hg, and the NO content was (38.91±8.67) nmol/mL. In group C, the apoptosis rate was 27.43% ± 0.03%, the APPL1 expression level was 2.03 ± 0.22, the LV -dP/dt was (559.38 ± 97.37) mm Hg/ s, the LVEDP was (12.55 ± 2.87) mm Hg, and the NO content was (138.79 ± 7.23) nmol/ mL. For the above mentioned parameters, there was significant difference between group A and group B (P lt; 0.05), and between group B and group C (P lt; 0.05). Conclusion BMSCs transplantation can improve the cardiac function of diabetic rats. Its possible mechanismmay be related to the activation of APPL1 signaling pathway and the increase of NO content.