[Experimental study on mechanical properties of decellularized porcine aortic valve and effects of precoating methods of biological scaffold on histocompatibility].

OBJECTIVE: To observe the mechanical properties of decellularized porcine aortic valve, and to explore the effects of precoating methods of biological scaffold on histocompatibility. METHODS: Fresh porcine aortic valves were decellularized using trypsin, TritonX-100 and nuclease. Treated valves were evaluated by light microscopy, scanning electron microscopy (SEM) and mechanical test. Three groups of scaffold were precoated with phosphate buffered saline (PBS), poly-L-lysine (PLL) and fetal bovine serum (FBS) respectively. Myofibroblasts were seeded onto each scaffold. Light and electron microscopic observation was performed and MTT test was used to examine efficiency of cell attachment. RESULTS: HE stain and SEM showed that cells were almost absent in the treated leaflet. The wave-like collagen together with the whole three-dimensional structure was maintained. Compared with normal valves, the Max-load, Max-stress and elastic modulus decreased while the Max-strain increased (P<0.05). The result of MTT test showed more cells were attached on the valves treated with FBS compared to the other two groups. Histological investigations also confirm that the high degree of cell attachment on the valves precoated with FBS (F=129.26, P=0.000). CONCLUSIONS: Enzyme combined with detergent and nucleases can remove cells from porcine aortic valves. Meanwhile the mechanical properties of these valves may be altered. Precoating porcine aortic valve with FBS is an effective method to improve cell attachment, growth and increasing.

[Association of polymorphism of Met764Thr locus allele in ADAM33 gene with bronchial asthma and lung function of asthmatic subjects].

OBJECTIVE: To analyze the association of the polymorphism of Met764Thr with bronchial asthma and lung function of asthmatic subjects of Han nationality in Southern China. METHODS: In 164 unrelated patients with asthma and 112 unrelated healthy controls, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing were used to determine polymorphism of Met764Thr locus allele in ADAM33 gene. The clinical indexes associated with lung function (FVC%, FEV(1)%) were compared among the three genotypes (Met764/Met764, Met764/Thr764, Thr764/Thr764) in asthmatic subjects. RESULTS: No significant difference was found in the allele (Met764, Thr 764) frequency among populations in UK, US, German, Korean, and Southern China (chi(2) = 6.77, P > 0.05). The frequencies of the genotypes (Met764/Met764, Met764/Thr764, Thr764/Thr764) were respectively 78.7% (129), 18.3% (30), 3.0% (5) in 164 asthmatic subjects and respectively 91.1% (102), 6.3% (7), 2.7% (3) in the 112 controls. There was a significant difference in the distributions of the genotypes (Met764/Met764, Met764/Thr764, Thr764/Thr764) between asthmatic subjects and controls (chi(2) = 8.46, P < 0.05). The frequencies of alleles (Thr764) were respectively 12.2% in the asthmatic subjects and 5.8% in the controls. Significant difference was observed in the allele (Met764, Thr 764) frequency between the two groups (chi(2) = 6.27, P < 0.05). The presence of Thr764 allele of ADAM33 gene was found to be a greater risk factor in asthmatic subjects than in controls. The odds ratio (OR) of Met764/Thr764 and Met764/Thr764 + Thr764/Thr764 were 3.389 (1.430 - 8.030), 2.767 (1.308 - 5.854), respectively. When compared with Met764/Met764 genotype, all P < 0.05. There was a significant decrease in the FVC% and FEV(1)% levels of Met764/Thr764, Thr764/Thr764 and Met764/Met764 genotype. CONCLUSIONS: These results suggest that Met764Thr locus genetic polymorphism is associated with susceptibility of asthma and clinical indexes of lung function of asthmatic subjects of Han nationality in Southern China.

[Application of transforming growth factor-beta(1) on construction of tissue engineering heart valves: experimental in vitro].

OBJECTIVE: To investigate the effects of transforming growth factor-beta1 (TGF-beta1) on construction of tissue engineering heart valves (TEHV). METHODS: Fresh porcine aortic valves were decellularized with trypsinase and detergent Triton X-100. Myofibroblasts were obtained from rat thoracic aorta, cultured, transfected with the vector containing TGF-beta1 gene-plasmid pcDNA3.0/TGF-beta1 mediated by lipofectamine 2,000 after 48 hours, screened by G418 for for 3 weeks. Decellularized valves were divided into 3 groups: Group A, seeded with the transfected myofibroblasts and cultured in medium without TGF-beta1, Group B, seeded with the transfected myofibroblasts and cultured in medium with TGF-beta1 10 ng/ml, and Group C, seeded with non-transfected myofibroblasts and cultured in medium without TGF-beta1. Hematoxylin-eosin staining and transmission electron microscopy were performed to observe the cell proliferation. DNA contents were measured. Hydroxyproline content was measured so as to indirectly test the collagen production. AGS-J mechanical testing instrument was used to test the mechanical properties of the strips of valves. RESULTS: Immunohistological investigation showed instant TGF-beta1 expression in the myofibroblasts 48 h after the transfection and stable TGF-beta1 expression 4 weeks later. Morphological examination showed that the myofibroblasts in Groups A and B were connected to one another closely with abundant extracellular matrix in the valves. The DNA contents of Groups A and B were (0.126 +/- 0.013) per thousand, and (0.109 +/- 0.004) per thousand, both significantly higher than that of Group [(0.089 +/- 0.011) per thousand, both P < 0.011], with a significant difference between Groups A and B (P < 0.05). The hydroxyproline content of Groups A and B were (5.83 +/- 0.67) per thousand and (5.02 +/- 0.40) per thousand, both significantly higher than that of Group C [(4.34 +/- 0.47) per thousand, both P < 0.05], with a significant difference between Groups A and B (P < 0.05). The maximum load of Groups A and B were (13.4 +/- 1.0) N and (11.7 +/- 1.4) N respectively, both significantly higher than that of Group C [(10.0 +/- 1.1) N, both P < 0.05], with a significant difference between Groups A and B (P < 0.05). CONCLUSION: TGF-beta1 is an important and effective bioactive factor for cell proliferation and extracellular matrix growth of heart valve. It is of great value for constructing TEHV in vitro.

[Association between ADAM33 gene polymorphism and bronchial asthma in South China Han population].

OBJECTIVE: To investigate the association between the polymorphism of T(1) locus allele in ADAM33 gene and bronchial asthma in South China Han population. METHODS: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing were used to determine the polymorphism of T(1) locus allele in ADAM33 gene in 160 unrelated patients with asthma and 95 unrelated healthy controls from South China Han population. RESULTS: No significant difference was found in T(1) locus allele distribution frequency in populations of UK, US, Germany, Korea, and South China (Chi(2)=9.085, P=0.109). The frequencies of the genotypes (TT, TC, CC) were 80.6% (n=129), 16.9% (n=27) and 2.5% (n=4) in the 160 asthmatic patients and 94.7% (n=90), 3.2% (n=3) and 2.1% (n= 2) in the 95 controls, respectively, showing a significant difference in the distribution of the genotypes (TT, TC, CC ) between asthmatic patients and healthy controls (Chi(2)=10.955, P<0.05). The frequencies of the alleles (T, C) were 0.891 and 0.109 in the asthmatic patients and 0.963 and 0.037 in the controls, respectively, showing also a significant difference in the allele frequency between them (Chi square =8.299, P<0.05). The presence of C allele of ADAM33 gene T1 locus was found to be a greater risk factor in asthmatic patients than in the healthy controls. The odds ratio (OR) of TC and TC+CC were 6.279 (1.849-21.328) and 4.326 (1.620-11.550), respectively, with P value of 0.001 and 0.002, respectively, in comparison with TT genotype. CONCLUSION: The polymorphism of T(1) locus allele in ADAM33 gene is associated with the susceptibility to asthma in South China Han population.