[Establishment of 43-plex SNP Typing System and Its Forensic Application].

OBJECTIVES: To evaluate the application of 43-plex SNP typing system in forensic science. METHODS: The typing of 43 SNP loci in 123 unrelated Han individuals from East China was detected by MALDI-TOF-MS. The application value of 43-plex SNP typing system was assessed according to the forensic parameters of population genetics. RESULTS: All the 43 SNP loci of 123 individuals showed no significant departure from Hardy-Weinberg equilibrium （P>0.05）. Excepted rs1355366, rs2270529, rs10776839 and rs938283, there were 39 SNP loci had minor allele frequencies （MAF）, which were greater than 0.25. Among the 25 loci MAFs, 24 ranged from 0.4 to 0.5, while 3 were close to 0.4. The DP, CDP, PIC, Ho, PEtrio and PEduo of the 43 SNP loci were 0.290 1-0.654 4, 1-9.8×10⁻¹¹, 0.170 8-0.500 0, 0.155 7-0.593 5, 0.085 4-0.250 0 and 0.014 6-0.125 0, respectively. The CPEtrio and CPEduo were 0.999 986 and 0.992 436 1, respectively. CONCLUSIONS: The 43-plex SNP typing system in present study shows a high polymorphism, which can be an effective supplement and verification for traditional STR genetic markers. It also can be used with other commercial kits for the forensic paternity testing and individual identification.

In vivo growth suppression of CT-26 mouse colorectal cancer cells by adenovirus-expressed small hairpin RNA specifically targeting thymosin beta-4 mRNA.

Thymosin beta-4 (Tß4) is known to be involved in tumorigenesis. Overexpression of this polypeptide has been observed in a wide variety of cancers, including colorectal carcinoma (CRC). Accordingly, Tß4 has been proposed to be a novel therapeutic target for CRC, especially in its metastatic form. Although in vitro tumor-suppressive effects of Tß4 gene silencing mediated by small hairpin RNA (shRNA) have already been demonstrated, the in vivo efficacy of such an approach has not yet been reported. Herein, we demonstrated that infection with recombinant adenovirus expressing an shRNA targeting Tß4 markedly reduced the growth of and robustly induced apoptosis in CT-26 mouse CRC cells in culture. Additionally, tumors grown in nude mice from the CT-26 cells whose Tß4 expression already been downregulated by virus infection were also drastically reduced. Most importantly, significant growth arrest of tumors derived from the parental CT-26 cells was observed after multiple intratumoral injections of these viruses. Together, our results show for the first time that in vivo silencing of Tß4 expression by its shRNA generated after adenoviral infection can suppress CRC growth. These results further demonstrate the feasibility of treating CRC by a Tß4 knockdown gene therapeutic approach.

Efficacy of Tiopronin in treatment of severe non-alcoholic fatty liver disease.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is a clinicopathologic syndrome of which the main feature is diffuse macrovesicular hepatic steatosis caused by deposition of excessive free fatty acid and triglyceride in liver parenchyma. AIM: To observe the efficacy of Tiopronin in treatment of severe nonalcoholic fatty liver disease (NAFLD). PATIENTS AND METHODS: 30 patients with severe NAFLD were treated with Tiopronin for 3 months. 30 healthy people were selected as control. The body mass index (BMI) and plasma levels of endotoxin (ET), leptin, IL-6 and IL-8 were measured before and after treatment. RESULTS: The serum levels of ET, leptin, IL-6 and IL-8 in severe NAFLD group were significantly higher than those in control group (p < 0.05). After treatment with Tiopronin, these indexes were significantly lower than before (p < 0.05). CONCLUSIONS: The intestinal endotoxemia (IETM) occurs in patients with severe NAFLD. Leptin, IL-6 and IL-8 play important roles in pathogenesis of NAFLD. Tiopronin can reduce the levels of ET, leptin, IL-6 and IL-8 for treatment of NAFLD.

Thymosin beta4 triggers an epithelial-mesenchymal transition in colorectal carcinoma by upregulating integrin-linked kinase.

The epithelial-mesenchymal transition (EMT) is crucial for the invasion and metastasis of many epithelial tumors including colorectal carcinoma (CRC). In the present study, a scattering and fibroblastic morphology with reduced intercellular contacts was found in the SW480 colon cancer cells overexpressing the gene encoding thymosin beta4 (Tbeta4), which was accompanied by a loss of E-cadherin as well as a cytosolic accumulation of beta-catenin, two most prominent markers of EMT. Whereas E-cadherin downregulation was likely to be accounted by a ZEB1-mediated transcriptional repression, the accumulation of beta-catenin was a result of glycogen synthase kinase-3beta inactivation mediated by integrin-linked kinase (ILK) and/or its downstream effector, Akt. Intriguingly, ILK upregulation in Tbeta4-overexpressing SW480 cells seemed to be attributed mainly to a stabilization of this kinase by complexing with particularly interesting new Cys-His protein (PINCH) more efficiently. In the meantime, a strong correlation between the expression levels of Tbeta4, ILK and E-cadherin in CRC patients was also revealed by immunohistochemical analysis. Taken together, these data suggest a novel role of Tbeta4 in promoting CRC progression by inducing an EMT in tumor cells via upregulating ILK and consequentially its signal transduction.

Lymphangiomyomatosis and angiomyolipoma: closely related entities characterized by hamartomatous proliferation of HMB-45-positive smooth muscle.

Angiomyolipoma is a hamartomatous condition which can occur as a component of the tuberous sclerosis complex. Lymphangiomyomatosis, another hamartomatous lesion occurring predominantly in the lungs, has long been suspected to be related to angiomyolipoma and tuberous sclerosis because of occasional clinical associations. We undertook this study to provide further support for the close relationship between these two entities. Five cases of lymphangiomyomatosis and 20 case of angiomyolipoma were retrieved for histological review and immunohistochemical studies. The antibodies used were anti-muscle specific actin (HHF-35), anti-desmin (D33) and anti-melanoma (HMB-45). Lesions featuring smooth muscle proliferation were used as controls. The proliferated smooth muscle cells in both lymphangiomyomatosis and angiomyolipoma were much plumper and paler or even clear, when compared with the deeply eosinophilic cytoplasm of the normal spindly smooth muscle cells and those of leiomyomas. Their nuclei were round to oval and pale rather than elongated and dark. Cells with bizarre nuclei were commoner in angiomyolipoma (18/20 cases) than lymphangiomyomatosis (1/5). In 12 cases of angiomyolipoma there were foci indistinguishable from lymphangiomyomatosis, i.e. plump spindle cells arranged in short fascicles around ramifying endothelium-lined spaces. All five cases of lymphangiomyomatosis stained for muscle-specific actin, desmin and HMB-45. For angiomyolipomas, the positivity rates for these markers were: 20/20, 17/20 and 18/20, respectively, including one case that was negative for both desmin and HMB-45. The various smooth muscle proliferations and tumours selected as controls were uniformly HMB-45 negative. The distinctive cytological features, morphological overlap and immunophenotypic profile all support a close relationship between lymphangiomyomatosis and angiomyolipoma, which probably represent different morphological manifestations of hamartomatous proliferation of a peculiar form of HMB-45-positive smooth muscle.