Objective To construct recombinant adenovirus vector containing human transforming growth factor beta 3 (TGF-β3), which was transfected into marrow mesenchymal stem cells(MSCs) and to observe its expression. Methods The cDNA TGF-β3 was intergraded into the shuttle vector of pAdTrack-CMV and recombinated with adenovirus skeleton vector pAdEasy-1 by homologous recombination. Then the product was transfected into package cell HEK293 by lipofedtamine and the recombinant adenovirus expressing the TGF-β3genewas generated. The rabbit’s MSCs were isolated, cultivated, purified, and then transfected with recombinant adenovirus containing the TGF-β3 gene. The green fluorescence protein expression was observed after 10 days, and the TGF-β3 expression was observed in MSCs transfected by recombinated adenovirus with TGF-β3 gene after 4 days. Results PCR showed that TGF-β3 cDNA was inserted into the recombinantadenoviral plasmid. The recombinant virus vectors with TGF-β3 gene were collected by the packaging HEK293 cells. The fusion rate of MSCs was 70%-80% with an intensive adhesion and uninform shape after the cultured 10th day. Fluorescent microscopy and immunocytochemistry demonstrated that TGF-β3 was expressed in MSCs. Conclusion Successful construction of human TGF-β3 recombinant adenovirus and its expression in MSCs provide a basis of research for the gene therapy of wound healing.
【Abstract】 Objective To investigate the effects on forming of hypertrophic scar after BMSCs infected with adenovirus carrying TGF-β3c2s2 were transplanted into the wound of animal scar model. Methods The third passage of rabbit’ s BMSCs were infected with 150 mutiple infection, and were cultured 24 hours. The concentration of the BMSCs infected with recombinant adenovirus containing the TGF-β3c2s2 gene was 1×105cell/mL. The purified and evaporated recombinant adenovirus grains containing the TGF-β3c2s2 gene were diluted by DMEM/F12 (without FBS) to 1×108 pfu/mL. The animal scar model of the standard Japanese big ear rabbit was establ ished. Eighty wounds were generated on the gastroside of ear and were randomized to 4 groups in each rabbit, which were divided into 3 control groups (A: control, B: Ad-TGF-β3c2s2, C: BMSCs) and 1 experimental group (D: BMSCs/Ad-TGF-β3c2s2). Then the wounds were tranplanted with cells. On 45 days and 90 days after wounded, thicknessand hardness of scars were measured with color ultrasound diagnostic unit and especial measurement for skin and scar hardness. On 21, 45 and 90 days, three specimens were harvested respectively for further histological study. Results The wound of groups A, B, C gradually formed the different degree scars after epithel ial ization. The hyperplasty of scars reached peak on 45 days after wounded and lasted about 90 days. There was no prominent scar formed in group D during the whole observed procedure. Thickness and hardness of scar of group D and group E were approximate on 45 days and 90 days. Thickness and hardness of scar of groups A, B and C were lower than those of group D (P lt; 0.01), and group B showed more lower than group A and group C (P lt; 0.01). Disorder structure and overlapping arrangement, enlargement collagen fibers were showed in the HE histological sections of the scars of groups A, C. The structure of the scars of groups B, C were similar to Group E. The constitutionsof groups A, B, C, D on 90 days resembled to each one on 45 days. In section of immunohistochemistry after wounded on21 days and 45 days, positive expressions of BrdU in nucleus of Groups C, D were observed. Negative expressions of BrdU in Groups A, B, E were showed. Conclusion BMSCs with Ad-TGF-β3c2s2 gene transplanted into wound could inhibit the forming of hypertrophic scar.
【Abstract】 Objective To investigate the role of myosin l ight chain (Myl) in myogenesis in vitro. Methods The extraocular muscle, diaphragm and gastrocnemius muscle myoblasts (eMb, dMb and gMb) were isolated and purified from 12 3-week-old C57BL/6 mice by using the enzyme digestion and Preplate technique, and then were subcultivated. The Myl expression in Mb was detected by RT-PCR and Western blot analysis; the Mb prol iferation activity was tested by methylene blue assay, and the myotube formation was observed. After anti-Myl antibody (1, 2, 3, 8, 16 ng/mL) was induced in the Mb culture (experimental group), the abil ity of prol iferation of myoblasts and the myotube formation were identified. Meanwhile, the Mb which was cultured without anti-Myl antibody was indentified as the control group. Results The results of RT-PCR and Western blot analysis showed that Myl1 and Myl4 mRNA and Myl protein were expressed in eMb, dMb and gMb at 24 hours after seeding, and their expression level were lower in eMb than in dMb and gMb (P lt; 0.01), and the latter two did not show any significant difference (P gt; 0.05). Myl2 and Myl3 mRNA was not detected in these three myoblasts. The prol iferation assay showed that the eMb prol iferated faster as compared with dMb and gMb (P lt; 0.01). eMb began to yield myotubes at 40 hours after seeding and dMb and gMb at 16 hours after seeding. At 6 days, the number of myotubes derived from eMb was (137.2 ± 24.5)/ field, which was significantly larger than that of myotubes from dMb [(47.6 ± 15.5) / field ] and gMb [(39.8 ± 5.1) field ] (P lt; 0.01). There was not statistically significant difference between the latter two groups (P gt; 0.05). After the antibody treatment, the absorbency values of the eMb, dMb and gMb in the experimental groups at each antibody concentration point were significantly higher than those in the corresponding control groups (P lt; 0.05), and the dose-dependent way was performed.The numbers of myotubes from dMb at 16 hours were (48.2 ± 7.1)/ well in the experimental group and (23.4 ± 4.9)/ well in the control group, and at 6 days were (40.6 ± 10.2)/ field in the experimental group and (63.1 ± 6.1)/ field in the control group.There was statistically significant difference between the experimental and control groups (P lt; 0.01). Conclusion Myl may play a role in myogenesis through the negative effect on the myoblast prol iferation.
Objective To investigate the effectiveness of autologous nano-fat mixed granule fat transplantation in the treatment of facial soft tissue dysplasia in children with mild hemifacial microsomia (HFM). Methods A total of 24 children with Pruzansky-Kaban type Ⅰ HFM were admitted between July 2016 and December 2020. Among them, 12 children were treated with autologous nano-fat mixed granule fat (1∶1) transplantation as study group and 12 with autologous granule fat transplantation as control group. There was no significant difference in gender, age, and affected side between groups (P>0.05). The child’s face was divided into region Ⅰ(mental point-mandibular angle-oral angle), region Ⅱ (mandibular angle-earlobe-lateral border of the nasal alar-oral angle), region Ⅲ (earlobe-lateral border of the nasal alar-inner canthus-foot of ear wheel). Based on the preoperative maxillofacial CT scan+three-dimensional reconstruction data, the differences of soft tissue volume between the healthy and affected sides in the 3 regions were calculated by Mimics software to determine the amount of autologous fat extraction or grafting. The distances between mandibular angle and oral angle (mandibular angle-oral angle), between mandibular angle and outer canthus (mandibular angle-outer canthus), and between earlobe and lateral border of the nasal alar (earlobe-lateral border of the nasal alar), and the soft tissue volumes in regions Ⅰ, Ⅱ, and Ⅲ of healthy and affected sides were measured at 1 day before operation and 1 year after operation. The differences between healthy and affected sides of the above indicators were calculated as the evaluation indexes for statistical analysis. At 1 year after operation, the parents, the surgeons, and the nurses in the operation group made a self-assessment of satisfaction according to the frontal photos of the children before and after operation. Results The study group and the control group were injected with (28.61±8.59) and (29.33±8.08) mL of fat respectively, with no significant difference (t=0.204, P=0.840). After injection, 1 child in the control group had a little subcutaneous induration, and no related complications occurred in the others. All children in both groups were followed up 1 year to 1 year and 6 months, with an average of 1 year and 4 months in the study group and 1 year and 3 months in the control group. At 1 year after operation, the asymmetry of the healthy and affected sides improved in both groups; the satisfactions of parents, surgeons, and nurses in the study group were all 100% (12/12), while those of the control group were 100% (12/12), 83% (10/12), and 92% (11/12), respectively. The differences between healthy and affected sides in mandibular angle-oral angle, mandibular angle-outer canthus, earlobe-lateral border of the nasal alar, and the soft tissue volume in 3 regions of the two groups after operation were significantly smaller than those before operation (P<0.05). There was no significant difference in the above indexes between the two groups before operation (P>0.05). After operation, all indexes were significantly lower in study group than in control group (P<0.05). Conclusion Autologous nano-fat mixed granule fat transplantation and autologous granule fat transplantation can both improve the facial soft tissue dysplasia in children with mild HFM, and the former is better than the latter.