[Molecular analysis of gene mutations in eight patients with Glanzmann's thrombasthenia].

Objective: To analyze the gene sequencing in eight patients with Glanzmann's thromboasthenia(GT), and combined with clinical manifestations and laboratory findings to investigate the molecular mechanism of GT. Methods: Eight patients who were diagnosed as GT based on platelet aggregation test and flow cytometry were enrolled, as well as 4 pedigrees. Next-generation sequencing was used to analyze all the exons and flanking sequences of αⅡ band ß3 gene and also platelet-type bleeding disorders related genes. Gene polymorphism was excluded by retrievaling HGMD and PubMed databases and relative literature. Mutations were confirmed by sanger sequencing. Results: All the eight patients had relatively normal platelet counts and coagulation profiles. But their platelet response to ADP was impaired, and their platelet response to ristocetin was relatively normal. Flow cytometry showed that of the 8 patients, platelet surface αⅡb/ß3 was lower than 5% of the normal value in 5 cases, and in 2 cases was 5% to 20% of normal value, and in 1 case there was no significant platelet surface αⅡb/ß3 reduction compared with normal level. Gene analysis revealed that five mutations in ITGA2B gene were identified, including c. 1750C>T(p.Arg584Ter), c.1882C>T(p.Arg628Ter), c.814G>C(p.Val272Leu), c.2333A>C(p.Gln778Pro), c.432G>A(p.Trp144Ter). Six mutations in ITGB3 gene, including c. 719G>A(p.Arg240Gln), c.2248C>T(p.Arg750Ter), c.1495T>C(p.Cys499Arg), c.1728delC(p.Ser577ProfsTer92), c.877C>T(p.Gln293Ter), c. 1260G>A were identified. In addition, mutations in genes such as RUNX1, HPS4, MYH9, ACTN1, HPS3 and SETBP1 were identified in patients with GT. Conclusions: Rather than homozygous mutations, heterozygous mutations, especially compound heterozygous mutations, are more common in patients with GT. The pathogenesis of GT may relate to gene mutations such as RUNX1 in addition to the ITGA2B gene and the ITGB3 gene.

Analysis of ODC and c-myc gene expression in hepatocellular carcinoma by in situ hybridization and immunohistochemistry.

We quantitated mRNA and protein for ornithine decarboxylase (ODC) and c-myc in formalin-fixed liver sections from 25 specimens of hepatocellular carcinoma (HCC) and seven normal livers by a non-radiolabeled in situ hybridization technique and immunohistochemistry. This non-radioactive in situ hybridization technique was highly specific, with virtually no background, and permitted quantitative analysis based on optical density. Reaction products were quantitated with computer-assisted microdensitometry. Samples were classified as normal, adjacent uninvolved, cirrhosis, well-differentiated HCC, and poorly-differentiated HCC. There was a progressive increase in all four parameters measured, ODC mRNA and protein, and c-myc mRNA and protein, from normal, to adjacent uninvolved liver, to cirrhosis, to well-differentiated HCC, to poorly-differentiated HCC. The sole exception was that ODC mRNA was lowest in cirrhosis. The patterns of ODC and c-myc gene expression are similar in HCC. The quantitative detection of ODC mRNA, c-myc mRNA, and their protein products in hepatocellular carcinoma and cirrhosis by in situ hybridization and immunohistochemical techniques may have a potential role in the study of hepatocarcinogenesis and in the diagnosis of hepatocellular carcinoma.

Detection of ornithine decarboxylase messenger RNA in human hepatocellular carcinoma by in situ hybridization.

Ornithine decarboxylase (ODC) has been shown by biochemical analysis, to be important for cell proliferation and carcinogenesis in a variety of tissues, including the liver. We detected messenger RNA (mRNA) specific for the enzyme ODC in 18 patients with hepatocellular carcinoma by an in situ hybridization technique using a radiolabelled ODC probe on formalin-fixed liver specimens. Adjacent uninvolved liver tissues were used as controls. Among the adjacent uninvolved liver tissues, five showed evidence of cirrhosis. Poorly differentiated hepatocellular carcinoma has significantly higher levels of ODC mRNA than does well-differentiated hepatocellular carcinoma, which in turn has a significantly higher ODC mRNA level than adjacent uninvolved liver tissues; tissues showing evidence of cirrhosis, on the other hand, had a significantly lower ODC mRNA level than adjacent uninvolved liver tissue. This pattern of ODC gene expression in hepatocellular carcinoma is similar to the pattern of expression of other oncogenes in liver tumours. The quantitative detection of ODC mRNA in hepatocellular carcinoma by in situ hybridization may help elucidate the potential role of ODC in hepatocarcinogenesis.

[Separation and identification of main medicinal saponin components from mass cell cultures of Panax notoginseng (Burk) F. H. Chen].

Five main saponins were separated from the cell cultures of Panax notoginseng (Burk) F. H. Chen. They were sanchinoside Rb1, Re, R1, Rg1 and Rh1 identified by TLC, HPLC, IR, M. P., 13CNMR and EI-MS. Saponin components of the cell cultures were almost the same as those of the cultivated plants. But the content of saponins was different between the cell cultures and the cultivated plants. Saponin extraction and separation procedure suitable for the cell cultures has to be different from that for the cultivated plants.

Establishment of Panax notoginseng tissue culture.

Calli were induced from Panax notoginseng roots, tubers, stems, petioles, leaves, and flower buds, and preliminary identification of medicinal compounds in the callus was carried out by TLC, using subcultured sixth-generation calli. The results demonstrated that callus was capable of synthesizing saponins, including ginsenosides Rb1, Rg1, and Rh1. The saponin content (5.33%, D.W.) in the callus was higher than that in intact plants (4.25%, D.W.). Callus also produced a larger amount of sapogenins than intact plants. Callus growth rate was 54.0 mg D.W./liter/day. Callus induced from stems was superior in terms of growth, saponin and sapogenin contents, and saponin composition. MS medium proved optimal for growth of callus induced from stems. The optimum pH was about 5.8 and the optimum temperature about 26 degrees C. Light had little stimulatory effect on callus growth.