Objective To investigate the possibility of differentiation and spermiogenesis of spermatocytes under in vitro condition. Methods Testis biopsy was done in 11 cases with non-obstructive azoospermia.Cells were prepared from 9 samples with spermatocytes and cultured in medium containing follicle stimulating hormone 50 U/L and testosterone 1 μmol/L.Sperms and cells of other types were counted and the proportion of every cell type was calculated before and 24 hours after culture.Flow cytometry was conducted before and 24 hours after culture in 2 cases to analyze the ploidy of the cells. Results The proportion of sperm in 9 samples was ( 17.7 ± 8.9 ) ％ before culture and ( 25.6 ± 10.3 ) ％ after culture ( P =0.004).Sperm increased significantly after culture.Flow cytometry demonstrated that the diagram of 4 n,2 n and 1 n converted to 2 n and broader 1 n. Conclusion Meiosis of spermatocytes and the transformation of spermatid into sperm could arise in in vitro culture.

OBJECTIVETo detect putative suppressor loci involved in tumor progressing or metastases.

METHODSThirty microsatellite marker primers were employed to amply the corresponding loci of the genome DNA from 83 patients with sporadic colorectal cancer. The PCR products were electrophoresed on a 377 PRISM sequencer and the fluorescent signals were analyzed by Genotyper and Genescan software.

RESULTSThe data were obtained from 24 loci, with an average LOH frequency of 15.16%. The LOH at D2S206 and D2S364 was more frequent than 30%, and was less than 20% at the rest loci. Significant difference was observed between the percentage of LOH and tumor staging or differentiation at D2S142 (2q24.1), D2S126 (2q35), D2S2211 (2p24.2), D2S305 (2p23.3). Occarrence of deletion at the later two loci was correlative.

CONCLUSIONSFrequent LOH was not observed at the loci around known mismatch repair genes on chr. 2. The region between D2S305 (2p23.3) and D2S2211 (2p24.2) deleted holistically, and was correlated to the stage and differentiation of tumor attended by D2S142 (2q24.1) and D2S126 (2q35) on 2q. It is suggested that unknown genes associated with tumor progressing or metastases reside in the two loci on 2q or the region on 2p.

Adult ; Aged ; Aged, 80 and over ; Cell Differentiation ; Chromosome Mapping ; Colorectal Neoplasms ; genetics ; pathology ; Female ; Humans ; Loss of Heterozygosity ; Male ; Microsatellite Repeats ; Middle Aged ; Neoplasm Metastasis

OBJECTIVETo evaluate and map the putative tumor suppressor loci on chromosome 5 involved in tumor progress or metastasis.

METHODSChromosome 5 of 83 patients with sporadic colorectal cancer was systemically screened. Fifteen microsatellite marker primers labeled with 3 different fluorescents were used to amplify the corresponding loci of the genome DNA. The PCR products were electrophoresed on a 377 PRISM sequencer and the fluorescent signals were analyzed with Genotyper and Genescan software.

RESULTSThe highest loss of heterozygosity (LOH) ratio was found at D5S416 (48.15%) on 5p and at D5S471 (38.71%) on 5q. The region (5q13.3 - 31), where D5S471 and 3 neighboring loci (D5S428, D5S2027 and D5S2115) reside, presented high frequent LOH.

CONCLUSIONThe deletion of APC, MCC, CTNNA1 and IL cluster in the 5q 13.3 - 31.1 area play important role in the tumorogenesis of colorectal cancer, and the expected existence of another novel tumor suppressor gene on 5p is possible.

Adult ; Aged ; Aged, 80 and over ; Alleles ; Chromosome Deletion ; Chromosomes, Human, Pair 5 ; Colorectal Neoplasms ; genetics ; Female ; Genes, Tumor Suppressor ; Humans ; Loss of Heterozygosity ; Male ; Microsatellite Repeats ; genetics ; Middle Aged

Objective To investigate the proliferative inhibition effect and mechanism of total saponins from marsdenia tenacissima on human liver cancer HepG2 cells. Methods Human liver cancer HpeG2 cells cultured conventionally were divided into the marsdenia tenacissima saponins A control group, the experimental group and the blank control group. The experimental group was treated with different mass concentrations of total saponins from marsdenia tenacissima (0.14, 0.29, 0.58, 1.15, 2.13 mg/ml), while the marsdenia tenacissima saponins A control group was treated with marsdenia tenacissima saponins A (1.0 mg/ml), and the blank control group was established stimultaneously. The methyl thiazolyl tetrazolium (MTT) method was used to detect the effect of total saponins from marsdenia tenacissima on the activity of HepG2 cells, and the cell morphology was observed by using inverted microscopy. The cell apoptosis rate was detected by using flow cytometry from Annexin V-FITC/PI staining. The expressions of bcl-2, bax and p53 were analyzed by using Western blot after the drug effect. Results The results of cell activity test showed that total saponins from marsdenia tenacissima could inhibit the proliferation of HepG2 cells at the concentration of 0.29, 0.58, 1.15, 2.13 mg/ml, which was positively correlated with the concentration. The half maximal inhibitory concentration (IC 50) of cell growth inhibition of total saponins from marsdenia tenacissima on HepG2 cells was 0.75 mg/ml. Under inverted microscopy, the adherent cells were significantly reduced, the cells fell off into clusters and the debris was increased after the effect of total saponins from marsdenia tenacissima. Moreover, flow cytometry showed that total saponins from marsdenia tenacissima could increase the late apoptosis rate of HepG2 cells (P< 0.01). Western blot showed that the expression of bcl-2 was 0.62±0.16, 0.31±0.15, 0.84±0.09 and 1.00±0.11 respectively in the experimental group (total saponins from marsdenia tenacissima 0.58 mg/ml and 2.13 mg/ml), the marsdenia tenacissima saponins A control group and the blank control group;the expression of bax protein was 0.75±0.10, 0.83±0.12, 1.00±0.14 and 0.15±0.02, respectively in the above four groups; the expression of p53 protein was 0.63±0.08, 0.78±0.11, 1.00±0.13 and 0.18±0.02 respectively in the above four groups. The protein expression of bcl-2 was decreased and the protein expressions of bax and p53 were increased in the experiment group, and there was a statistical difference between the experiment group and the blank control group (P< 0.05). Conclusions Total saponins from marsdenia tenacissima can upregulate the expression of bax by upregulating the expression of p53 gene, and inhibit the expression of bcl-2, which would cause the cascade reaction to induce the apoptosis of HepG2 cells. Its inhibitory effect can be realized through mitochondrial pathmay to induce apoptosis.

OBJECTIVETo analyze the loss of heterozygosity (LOH) of chromosome 20 in patients with sporadic colorectal cancer to identify additional loci involved in colorectal tumorigenesis.

METHODSPolymorphic microsatellite markers were analyzed in 83 colorectal cancer patients' tumor and normal DNA by PCR. PCR products were electrophoresed on an 377 DNA sequencer. Genescan 2.1 and Genotype 2.1 software were used in the LOH scanning and analysis. Comparisons between LOH frequency and clinicopathological data were performed by chi(2) test. P < 0.05 was considered statistically significant.

RESULTSThe average LOH frequency in the long arm, short arm and whole chromosome 20 was 21.1%, 26.7% and 22.8%, respectively. Chromosome 20 exhibited relatively high LOH frequency, particularly in the regions of 20p and 20q11.1-q13.1.

CONCLUSIONThere is notable genetic instability on chromosome 20 in sporadic colorectal carcinoma patients; that is, mutation on chromosome 20 is closely associated with sporadic colorectal carcinogenesis. Also, there may be tumor suppressor genes related to sporadic colorectal carcinoma near the region 20q11.1-q13.1.

Adult ; Aged ; Aged, 80 and over ; Chromosomes, Human, Pair 20 ; Colorectal Neoplasms ; genetics ; pathology ; Female ; Humans ; Loss of Heterozygosity ; Male ; Microsatellite Repeats ; Middle Aged