Objective Col I A1 antisense oligodeoxyneucleotide (ASODN) has inhibitory effect on collagen synthesis in cultured human hypertrophic scar fibroblasts. To investigate the effects of intralesional injection of Col I A1 ASODN on collagen synthesis in human hypertrophic scar transplanted nude mouse model. Methods The animal model of humanhypertrophic scar transplantation was established in the 60 BALB/c-nunu nude mice (specific pathogen free grade, weighing about 20 g, and aged 6-8 weeks) by transplanting hypertrophic scar without epidermis donated by the patients into the interscapular subcutaneous region on the back, with 1 piece each mouse. Fifty-eight succeed models mice were randomly divided into 3 groups in accordance with the contents of injection. In group A (n=20): 5 μL Col I A1 ASODN (3 mmol/L), 3 μL l iposome, and 92 μL Opti-MEM I; in group B (n=20): 3 μL l iposome and 97 μL Opti-MEM I; in group C (n=18): only 100 μL Opti-MEM I. The injection was every day in the first 2 weeks and once every other day thereafter. The scar specimens were harvested at 2, 4, and 6 weeks after injection, respectively and the hardness of the scar tissue was measured. The collagens type I and III in the scar were observed under polarized l ight microscope after sirius red staining. The ultrastructures of the scar tissues were also observed under transmission electronic microscope (TEM). Additionally, the Col I A1 mRNAs expression was determined by RT-PCR and the concentrations of Col I A1 protein were measured with ELISA method. Results Seventeen mice died after intralesional injection. Totally 40 specimens out of 41 mice were suitable for nucleic acid and protein study, including 14 in group A, 13 in group B, and 14 in group C. The hardness of scars showed no significant difference (P gt; 0.05) among 3 groups at 2 weeks after injection, whereas the hardness of scars in group A was significantly lower than those in groups B and C at 4 and 6 weeks (P lt; 0.05), and there was no significant difference between groups B and C (P gt; 0.05). The collagen staining showed the increase of collagentype III in all groups, especially in group A with a regular arrangement of collagen type I fibers. TEM observation indicated that there was degeneration of fibroblasts and better organization of collagen fibers in group A, and the structures of collagen fibers in all groups became orderly with time. The relative expressions of Col I A1 mRNA and the concentrations of Col I A1 protein at 2 and 4 weeks after injection were significant difference among 3 groups (P lt; 0.05), and they were significantly lower in group A than in groups B and C (P lt; 0.05) at 6 weeks after injection, but no significant difference was found between groups B and C (P gt; 0.05). Conclusion Intralesional injection of Col I A1 ASODN in the nude mice model with human hypertrophic scars can inhibit the expression of Col I A1 mRNA and collagen type I, which enhances the mature and softening of the scar tissue. In this process, l iposome shows some assistant effect.
To investigate the inhibitory effect of Col I A1 antisense ol igodeoxyneucleotide (ASODN) transfection mediated by cationic l iposome on Col I A1 expression in human hypertrophic scar fibroblasts. Methods Scar tissue was obtained from volunteer donor. Human hypertrophic scar fibroblasts were cultured by tissue block method. The cells at passage 4 were seeded in a 6 well cell culture plate at 32.25 × 104 cells/well, and then divided into 4 groups: group A, l iposomeand Col I A1 ASODN; group B, Col I A1 ASODN; group C, l iposome; group D, blank control. At 8 hours, 1, 2, 3 and 4 days after transfection, total RNA of the cells were extracted, the expression level of Col I A1 mRNA was detected by RT-PCR, the Col I A1 protein in ECM was extracted by pepsin-digestion method, its concentration was detected by ELISA method. Results Agarose gel electrophoresis detection of ampl ified products showed clear bands without occurrence of indistinct band, obvious primer dimmer and tailing phenomenon. Relative expression level of Col I A1 mRNA: at 8 hours after transfection, group A was less than groups B, C and D (P lt; 0.05), and groups B and C were less than group D (P lt; 0.05), and no significant difference was evident between group B and group C (Pgt; 0.05); at 1 day after transfection, groups A and B were less than groups C and D (P lt; 0.05), and there was no significant difference between group A and group B, and between group C and group D (P gt; 0.05 ); at 2 days after transfection, there were significant differences among four groups (P lt; 0.05); at 3 and 4 days after transfection, group A was less than groups B, C and D (P lt; 0.05), group B was less than groups C and D (P lt; 0.05), and no significant difference was evident between group C and group D (P gt; 0.05). Concentration of Col I protein: at 8 hours after transfection, group A was less than groups B, C and D (P lt; 0.05), groups B and C were less than group D (P lt; 0.05), and no significant difference was evident between group B and group C (P gt; 0.05); at 1 day after transfection, significant differences were evident among four groups (P lt; 0.05); at 2, 3 and 4 days after tranfection, groups A and B were less than groups C and D (P lt; 0.05), and no significant difference was evident between group A and group B (P gt; 0.05). Conclusion Col I A1 ASODN can inhibit mRNA and protein expression level of Col I A1. Cationic l iposome, as the carrier, can enhance the inhibition by facil itating the entry of ASODN into cells and introducing ASODN into cell nucleus.
To investigate the preventive effect of TGF-β1 neutral izing antibody on collagen production and adhesion formation of flexor tendon. Methods Tendon fibroblasts, epitenon tenocytes, and endotenon tenocytes were obtained from 6 New Zealand rabbit flexor tendons. Each cell culture was supplemented with 1 ng/mL of TGF-β along with increasing dose of TGF-β1 neutral izing antibody. Col I production was measured by enzyme-l inked immunoabsorbent assay after 3 days. Eighty-four adult New Zealand White rabbits forepaws underwent sharp transection of middle digit flexor digitorumprofundus and immediate repair. Then the rabbits were divided into three groups: the normal saline (NS group, n=36), 1.0 µg/ mL TGF-β1neutral izing antibody (1.0 µg/mL TGF-β1group, n=36) and 2.0 µg/mL TGF-β1 neutral izing antibody (2.0 µg/mL TGF-β1 group, n=12) were injected in tendon sheath respectively. Tendons were harvested at 4 and 8 weeks for biomechanics testing, histological evaluation and scanning electron microscope observation. Tendons were harvested at 1, 2, 4 and 8 weeks to determine the mRNA expression of TGF-β1 and Col I by in situ hybridization. Results ELISA exhibed that TGF-β1 enhanced Col I production and the neutral izing antibody significantly inhibited TGF-β1-induced Col I production in all 3 cell culture with a dose-dependent. At 4 and 8 weeks after operation the gl iding excursion of the tendon and the simulated active flexion in NS group were less than that of 1.0 µg/mL TGF-β1 group and 2.0 µ g/mL TGF-β1 group. There was significant difference between NS group and 1.0 µ g/mL TGF-β1 group, 2.0 µ g/mL TGF-β1 group (P lt; 0.05). The tendon anastomosis breaking strength showed no significant differences among three groups (P gt; 0.05). Scanning electron microscope and histological observation showed that collagen fibers arranged irregularly in NS group, but arranged regularly in 1.0 µ g/mL TGF-β1 group and 2.0 µ g/mL TGF-β1group at 4 and 8 weeks after operation. The in situ hybridization results revealed that TGF-β1 and Col I mRNA expression in 1.0 µ g/mL TGF-β1 group was lower than that in NS group at each time. There was significant difference between two groups (P lt; 0.05). Conclusion TGF-β1neutral izing antibody can inhibit the function of the TGF-β1 effectively and prevent adhesion formation after the flexor tendon injured and repaired.
Objective To locate sinoatrial node (SAN) in suckl ing pigs, to develop a rel iable method for isolation, purification and cultivation of SAN cells and to observe the compatibil ity of SAN cells and Col I fiber scaffold. Methods Five newborn purebred ChangBaiShan suckl ing pigs (male and female), aged less than 1-day-old and weighing 0.45-0.55 kg, wereused. Multi-channels electrophysiological recorder was appl ied to detect the original site of atrial waves. Primary SAN cells harvested from that area were cultured by the conventional culture method and the purification culture method including differential velocity adherent technique and 5-BrdU treatment, respectively. Atrial myocytes isolated from the left atrium underwent purified culture. Cell morphology, time of cell attachment, time of unicellular pulsation, and pulsation frequency were observed using inverted microscope. The purified cultured SAN cells (5 × 105 cells/mL) were co-cultured with prewetted Col I fiber scaffold for 5 days, and then the cells were observed by HE staining and scanning electron microscope (SEM). Results The atrial waves occurred firstly at the area of SAN. The purified cultured SAN cells were spindle, triangular, and irregular in morphology, and the spindle cells comprised the greatest proportion. Atrial myocytes were not spindle-shaped, but primarily triangular and irregular. The proportion of spindle cells in the conventional cultured SAN cells was decreased from 73.0% ± 2.9% in the purified cultured SAN cells, to 44.7% ± 2.3% (P lt; 0.01), and the proportion of irregular cells increased from 7.0% ± 1.7% in the purified cultrued SAN cells to 36.1% ± 2.6% (P lt; 0.01) . The proportion of the triangular cells in the purified and the conventional cultured SAN cells was 20.0% ± 2.1% and 19.2% ± 2.5%, respectively (P gt; 0.05). At 5 days after co-culture, HE staining displayed lots of SAN cells in Col I fiber scaffold, and SEM demonstrated conglobate adherence of the cells to the surface and lateral pore wall of scaffold, mutual connections of the cell processes, or attachment of cells to lateral pore wall of scaffold through pseudopodia. Conclusion With accurate SAN location, the purification culture method containing differential velocity adherent technique and 5-BrdU treatment can increase the proportion of spindle cells and is a rel iable method for the purification and cultivation of SAN cells. The SAN cells and Col I fiber scaffold have a good cellular compatibil ity.
Objective To examine the effects of alendronate (ALN) on IL-1β-stimulated chondrocyte of rabbit in vitro and on cartilage and subchondral bone in rabbit osteoarthritis (OA) induced by anterior cruciate l igament transection (ACLT). Methods The chondrocytes from articular surface of healthy 3-month-old Japanese White rabbits were obtained by the method of enzyme digestion and cultured in vitro. The third generation chondrocytes were assigned into three groups: thechondrocytes were cultured in DMEM medium with 10 ng/mL IL-1β for 2 days, subsequently with (ALN group, group A1) orwithout (IL-1β group, group B1) 1 × 10-6 mol/L ALN for 3 days; the chondrocytes in vacant group (group C1) were cultured in DMEM medium for 5 days. The expression of Col II and MMP-13 were analyzed by immunocytochemical staining observation and real time RT-PCR test. Another twenty-four 3-month-old male Japanese White rabbits were randomized into three groups (n=8 per group). The OA model was made by ACLT in ACLT+ALN group (group A2) and ACLT group (group B2); the joint cave was sutured after exposure of ACL in sham group (group C2). After 4 days, the rabbits of group A2 received the subcutaneous injection of ALN at a dosage of 10 μg/(kg·d) for 8 weeks. Rabbits of group B2 and C2 received equal normal sal ine treatment. After 8 weeks, the rabbits were executed. The macro-pathologic changes of right knee joints were observed, so were the histological changes of femoral condyles. Expression levels of Col II and MMP-13 were detected by immunohistochemical staining. The bone histomorphometry analysis was appl ied to subchondral bone of proximal tibia. Results In vitro, the Col II immunocytochemical staining showed intensely positive staining in group C1, and the intensity of staining was sl ightly decreased in group A1, but the intensity of Col II immunocytochemical staining was extremely lower in the group B1. The integrated absorbance (IA) value for Col II in group A1 was significantly higher than that of group B1 (P lt; 0.05), but there was no significant difference between group A1 and group C1 (P gt; 0.05). Immunocytochemical detection of MMP-13 showed intense staining in group B1, and the intensity of staining was sl ightly decreased in group A1, but no MMP-13 expression was detected in the group C1. The IA value for MMP-13 in group A1 was significantly lower than that of group B1 (P lt; 0.05), but significantly higher than that of group C1 (P lt; 0.05). The real time RT-PCR analysis showed significantly higher mRNA levels of Col II in group A1 than in group B1 (P lt; 0.05), but there was no significant difference between group A1 and group C1 (P gt; 0.05). The MMP-13 mRNA level of the chondrocytes in group A1 was significantly lower than that of group B1 (P lt; 0.05), but significantly higher than that of group C1 (P lt; 0.05). In vivo, the gross appearance of surface of knee joint showed that there was no ulcer in group C2, and there was some ulcers in group A2, but many and all layers ulcers in group B2. Mankin score of group A2 was significantly lowerthan that of group B2 (P lt; 0.05), but significantly higher than that of group C2 (P lt; 0.05). Immunohistochemical staining showed that Col II in articular cartilage was intensely staining in group C2, the intensity of staining was sl ightly decreased in group A2, and the intensity of Col II immunohistochemical staining was extremely low in group B2, but there was no significant difference between group A2 and group C2 (P gt; 0.05..........