P62/SQSTM1 mediates the autophagy-lysosome degradation of CDK2 protein undergoing PI3Kα/AKT T308 inhibition.

CDK2 forms a complex with cyclin A and cyclin E to promote the progress of cell cycle, but when cyclin A and cyclin E are dissociated from the complex and degraded by the ubiquitin proteasome pathway, the fate of the inactive CDK2 is unclear. In this study, we found that the inactive CDK2 protein was degraded by autophagy-lysosome pathway. In the classic model of G0/G1 phase arrest induced by serum starvation, we found that the mRNA level in CDK2 did not change but the protein level decreased. Subsequently, using PI3K and AKT inhibitors and gene knockout methods, it was found that CDK2 degradation was mediated by the inhibition of PI3Kα/AKTT308. In addition, P62/SQSTM1 was found to bind to the inactivated CDK2 protein to help it enter autophagy-lysosome degradation in a CTSB-dependent manner. Taken together, these results confirm that the PI3Kα/AKTT308 inhibition leads to degradation of CDK2 protein in the autophagy-lysosome pathway. These data reveal a new molecular mechanism of CDK2 protein degradation and provide a new strategy and method for regulating CDK2 protein.

Compound heterozygous variations in IARS1 cause recurrent liver failure and growth retardation in a Chinese patient: a case report.

BACKGROUND: Aminoacyl-tRNA synthetases (ARSs) are enzymes responsible for attaching amino acids to tRNA, which enables protein synthesis. Mutations in isoleucyl-tRNA synthetase (IARS1) have recently been reported to be a genetic cause for growth retardation, intellectual disability, muscular hypotonia, and infantile hepatopathy (GRIDHH). CASE PRESENTATION: In this study, we reported an additional case of compound heterozygous missense variations c.701 T > C (p.L234P) and c.1555C > T (p.R519C) in IARS1, which were identified using medical exome sequencing; c.701 T > C (p.L234P) was a novel variant, and c.1555C > T (p.R519C) was found in GnomAD. Unlike other reported patients, this individual presented prominently with recurrent liver failure, which led to her death at an early age of 19 months. She also had significant growth retardation, muscular hypotonia, chubby and flabby face, recurrent loose stools, and abnormal brain computed tomography (CT), while zinc deficiency and hearing loss were not present. Studies in zebrafish embryo modeling recapitulated some of the key phenotypic traits in embryo development, neurodevelopment, liver development, and myogenesis, demonstrating that these variations caused a loss of gene function in IARS1. CONCLUSIONS: We have found a novel mutation point c.701 T > C (p.L234P) in IARS1. Compound heterozygous mutations of c.701 T > C (p.L234P) and c.1555C > T (p.R519C) in IARS1 are pathogenic, which can cause GRIDHH in child.

[Application of Array-based Comparative Genomic Hybridization in the Prenatal Diagnosis of Fetal Chromosomal Aberration in Gravidas with Advanced Age].

OBJECTIVE: To evaluate the clinical application of array-based comparative genomic hybridization (a-CGH) in the prenatal diagnosis of fetal chromosomal aberrations in gravidas with advanced maternal age (AMA). METHODS: A total of 3 677 amniotic fluid samples from pregnant women who underwent amniocentesis for prenatal diagnosis solely due to AMA were selected. Array-CGH was performed on the Agilent CGX TM (8X60K) platform and the data were analyzed by the Genoglyphix software. RESULTS: The overall detection rate of chromosomal aberration was 2.04% (75/3677), with 53.33% (40/75) being aneuploidies, including 22 cases of trisomy-21, 5 cases of trisomy-18, 8 cases with XXY, 3 cases of XYY and 2 cases of mosaic monosomy X, 32.00% (24/75) being pathogenic copy number variations (pCNVs), including 19 cases of microdeletion and 5 cases of microduplication, with the fragment size ranging from 323 kb to 26 780 kb, and 14.67% (11/75) being likely pathogenic CNVs (lpCNVs), including 7 cases of microdeletion and 7 cases of microduplication, with the fragment size ranging from 358 kb to 16 873 kb. Besides, the detection rate of CNVs of unknown clinical significance (VUS) was 0.84% (31/3 677). The detection rate of aneuploidies increased significantly with increased maternal age ( P<0.05). However, there were no significant differences in the detection rate of p/lpCNVs among different maternal age groups ( P>0.05). CONCLUSION: Our findings suggest that, compared with traditional karyotype analysis, a-CGH not only detects aneuploidies, but also detect pathogenic CNVs, including microdeletion/microduplication syndromes. The detection rate of fetal aneuploidies was closely correlated to maternal age. However, no correlation was found between the detection rate of p/lpCNVs and maternal age.

[Application of Array-based Comparative Genomic Hybridization in Diagnostic Assessment of Abnormal Prenatal Serological Screening Results of Down's Syndrome].

OBJECTIVE: To explore the application of array-based comparative genomic hybridization (a-CGH) technology in the prenatal diagnostic assessment of abnormal serological prenatal screening results of Down's syndrome (DS). METHODS: A total of 3 578 amniotic fluid samples from pregnant women who underwent amniocentesis for prenatal diagnosis solely due to abnormal serological prenatal screening results were selected. The samples were categorized into 3 groups, 2 624 in the high-risk group, 662 in the borderline-risk group, and 292 in the abnormal multiple of median (MoM) group. a-CGH was performed on the Agilent CGX ™ (8×60K) platform and the data were analyzed by the Genoglyphix ® software. RESULTS: The overall detection rate of chromosomal abnormalities was 3.38% (121/3 578). Among the chromosomal abnormalities, 49.59% (60/121) was aneuploidies, 42.15% (51/121) was pathogenic copy number variants (pCNVs), and 8.26% (10/121) was likely pathogenic CNVs (lpCNVs). The detection rate of copy number variant of uncertain significance (VUS) was 1.03% (37/3 578). In the high-risk, the borderline-risk and the abnormal MoM groups, the detection rate of chromosomal abnormalities was 3.54% (93/2 624), 2.87% (19/662) and 3.08% (9/292), respectively; the detection rate of p/lp CNVs was 1.64% (43/2 624), 1.81% (12/662) and 2.05% (6/292), respectively; the detection rate of trisomy 21 and trisomy 18 was 1.37% (36/2 624), 0.76% (5/662) and 0.34% (1/292) in the three groups, respectively. There were no significant differences in all the detection rate among these groups ( P>0.05). One sample with X(51)/XYY(49) confirmed by fluorescence in situ hybridization (FISH) was misdiagnosed by a-CGH. CONCLUSION: Prenatal diagnosis with a-CGH is of great significance for reducing birth defects in pregnancies with abnormal serological prenatal screening results of DS. It can also be used to detect CNVs of microdeletion/microduplication syndromes.

MicroRNA-92b acts as an oncogene by targeting PTEN/AKT in NSCLC.

MicroRNAs can act as tumour suppressors or oncogenes by regulating cellular differentiation, proliferation and apoptosis, and the dysregulation of miRNA is involved in the occurrence and development of NSCLC. Here, we provided evidence that miR-92b as an oncogene in NSCLC by targeting PTEN/AKT. We found that miR-92b was up-regulated in human NSCLC tissues and cell lines. MiR-92b knockdown suppressed the NSCLC cells proliferation and migration in both in vivo and in vitro models. Conversely, miR-92b overexpression induced an aggressive phenotype. Moreover, miR-92b-mediated regulation of NSCLC cell proliferation and migration depended on binding to PTEN mRNA, which then led to the degradation of PTEN and activation of the downstream AKT signalling pathway. Overall, this study revealed the oncogenic roles of miR-92b in NSCLC by targeting PTEN/AKT, and provided novel insights for future treatments of NSCLC patients. SIGNIFICANCE OF THE STUDY: MiR-92b was up-regulated in human NSCLC tissues and cell lines. Our study demonstrated that miR-92b as an oncogene in NSCLC by targeting PTEN/AKT in both in vivo and in vitro models and provided novel insights for future treatments of NSCLC patients.

[Analysis of 21 723 Pregnant Women's First Trimester Screening for Down Syndrome in Sichuan Province].

OBJECTIVE: To compare the effect of different first-trimester screening programmes for Down syndrome in Sichuan Province. METHODS: We retrospectively collected the data of singleton pregnancies that were screened by serum biochemistry markers combined with nuchal translucency screening tests in the first trimester in Prenatal Diagnosis Center of West China Second University Hospital of Sichuan University from January 2011 to December 2017. The fetal chromosome results were obtained by amniocentesis or by telephone follow-up. The screening effect of maternal age, nuchal translucency thickness, maternal serum biochemistry markers and combined screening in the first trimester were analyzed. RESULTS: Among the 21 723 singleton pregnancies, 33 cases were diagnosed as Down syndrome, and 19 cases were diagnosed as trisomy 18 sex chromosome abnormalities were found in 4 cases, and other chromosome abnormalities were found in 8 cases. For the combined screening, the detection rate of Down syndrome was 72.73%, and the false positive rate was 2.49%; the detection rate of trisomy 18 syndrome was 73.68% with the false positive rate of 0.39%. With a 5% false positive rate, maternal age, nuchal translucency thickness, serum biochemistry markers and combined screening would respectively detect 15.15%, 57.58%, 60.61% and 87.88% of Down syndrome fetuses. CONCLUSION: Compared with the other three screening programmes, the combined screening can effectively screen fetuses with Down syndrome and other chromosomal abnormalities.

[Clinical Application Assessment of KaryoLite BoBs Combined with QF-PCR in the Detection of Products of Conception].

OBJECTIVE: To assess the accuracy and discuss the feasibility of KaryoLite bacterial artificial chromosome on beads (KL-BoBs) and quantitative fluorescent polymerase chain reaction (QF-PCR) in genetic testing of products of conception (POC) by comparing with the chromosomal microarray analysis (CMA) test results. METHODS: Eighty-one cases of abortion samples were collected in the prenatal diagnosis center of West China Second University Hospital in Sichuan University from May to August 2016,including 61 cases of placenta tissues,19 cases of fetal muscle tissues and 1 case of fetal liver tissue. KL-BoBs and QF-PCR were used to detect the samples. The results were compared with those of CMA test to evaluate the accuracy of KL-BoBs and QF-PCR. RESULTS: Of the 81 POC samples,the results of 70 samples tested by KL-BoBs was consistent with that of CMA. Among them,36 cases were normal karyotype and 34 cases were abnormal karyotypes (aneuploidy). Triploid could not been detected by KL-BoBs (the results were shown 2 cases were normal karyotype and 5 cases were aneuploidy),whereas CMA and QF-PCR could be detected. Copy number variation of small segments could not been detected by KL-Bobs. Four cases of copy number variationwere detected by CMA.Compared with CMA,the positive coincident rate of KL-BoBs combined with QF-PCR was 91.1% (41/45),the negative coincidence rate was 100% (36/36). The accuracy rate of KL-BoBs was 86.4% (70/81),the false positive was 0% and the false negative was 13.3% (6/45). Whereas both KL-BoBs and QF-PCR were simultaneously detected,the accuracy rate would be improved to 95.1% (77/81). CONCLUSION: The accuracy rate of KL-BoBs combined with QF-PCR was high for testing early pregnancy abortion tissue. It can be used as a first-tier test.

[Clinical Application of Chromosomal Microarray Analysis in Karyotyping with Uncertain Genomic Rearrangement].

OBJECTIVES: To apply chromosomal microarray analysis (CMA) in the diagnosis of karyotyping with uncertain genomic rearrangement. METHODS: We retrospectively reviewed 48 samples (34 samples of amniotic fluid, 14 samples of peripheral blood) of karyotype analyses with uncertain genomic rearrangement in patients admitted to our department from September 2014 to April 2016. The CMA results were compared with those of karyotyping. RESULTS: The 48 samples consisted of 13 samples with marker chromosomes, 19 samples with derivative chromosomes, and 16 samples with balanced translocation. Sixteen cases (33.33%) were detected with abnormalities by CMA. In the 32 samples with marker chromosomes or derivative chromosomes, 16 cases were detected with deletions or duplications (>5 Mb) by CMA, including 1 case 21-trisomy, 2 cases XYY syndrome and 3 cases microdeletion/ microduplication syndromes (22q11 duplication syndrome, Wolf-Hirschhorn syndrome and 15q26 overgrowth syndrome). In the 16 balanced translocation cases, all revealed negative results in CMA. CONCLUSIONS: CMA can confirm the karyotyping with uncertain genomic rearrangement and clarify its clinical significance.

[Application of Chromosomal Microarray Analysis for Chromosomal Abnormalities of Spontaneously Aborted Fetuses].

OBJECTIVE: To evaluate the clinical significance of chromosomal microarry analysis (CMA) for detection of chromosomal abnormalities in spontaneously aborted fetuses. METHODS: Chorionic villi samples from 431 spontaneously aborted fetuses were detected on the chromosomal abnormalities by CMA in our department form September 2014 to April 2016. RESULTS: The overall success rate of CMA was 100%,and 283 cases were detected with abnormalities (65.67%). Of these 283 cases with abnormal results,126 were common aneuploidies (trisomy 13,16,18,21,22 as well as X and Y aneuploidies) (44.52%),72 were uncommon aneuploidies (25.44%),10 were composited aneuploidies (3.53%),9 were partial aneuploidies (3.18%),29 were polyploidy (10.25%),4 were mosaicism (1.41%),31 were with multiple duplications and deletions (10.96%),and 2 were microduplication/microdeletion syndromes. CONCLUSION: CMA has great advantage for the detection of chromosomal abnormalities in spontaneously aborted fetuses comparing with fluorescence in situ hybridization (FISH). It is of great clinical significance for etiological diagnosis of spontaneous abortion and guidance on reproduction.

[Expression of USP9X in Non-small Cell Lung Cancer and Its Clinical Significance].

OBJECTIVE: To investigate the expression and clinical significance of ubiquitin-specific protease 9X (USP9X) protein in non-small cell lung cancer (NSCLC). METHODS: USP9X proteins were detected in 71 cases of NSCLC and 20 cases of benign pulmonary tissues by immunohistochemical staining. The correlation between USP9X expression and 51 NSCLC clinicopathological parameters as well as survival rates were indicated. RESULTS: Higher rate [69. 0% (49/71)] of the expression of USP9X was observed in NSCLC samples, compared with 20. 0% (4/20) in benign pulmonary tissues (P<0. 001). Furthermore, the expression of USP9X proteins was positively associated with both histological types and lymph node metastasis (P<0. 05). The survival analysis showed that the survival rate was lower in patients with positive expressions of USP9X than in patients with negative expressions (P< 0. 05). USP9X expression, together with histological types and TNM stage was an independent predictor for overall survival in the multivariate Cox regression model (P < 0. 05). CONCLUSION: Up-regulation of USP9X plays an important role in the invasion and progression of NSCLC and could be considered as a prognostic predictor for NSCLC.

[The influence of fibronectin on the formation of multi-cellular spheroid of ovarian cancer].

OBJECTIVE: To investigate the role of Fibronectin in the formation of multi-cellular spheroid of ovarian cancer and the integrin receptor involved in the process. METHODS: In vitro model of multi-cellular spheroid of SKOV3 was constructed by liguid overlay technique. The influence of fibronectin on the formation of the spheroid was observed. The gene expressions of potential integrin receptors were examined from the levels of mRNA and protein using real time reverse transcription PCR and Western-blot. RESULTS: Fibronctin stimulated the formation of multi-cellular spheroid of ovarian cancer larger than 250 microm. fibronectin suppressed the expression of subtype of integrin receptor ITGA5. CONCLUSION: Fibronectin can enhance the formation of multi-cellular spheroid of ovarian cancer. The subtype of integrin receptor ITGA5 is probably involved in the process.

[Expression of long non-coding RNA HOTAIR mRNA in ovarian cancer].

OBJECTIVE: To investigate the long non-coding RNA HOTAIR (long non-coding HOX antisense intergenic RNA) by examining HOTAIR expression levels in ovarian cancer tissue and normal ovarian tissue. METHODS: The mRNA of long non-coding RNA HOTAIR expressions were detected by real-time fluorescence quantitative PCR in ovarian cancer (44) and normal ovary tissues (14). RESULTS: The expression of HOTAIR in ovarian cancer tissue was higher than that in normal ovarian tissue (1.26 +/- 0.27 vs. 0.14 +/- 0.09, P < 0.05). The expression was statistically higher in poorly differentiated ovarian cancer than poorly-moderately, moderately-well, and well-differentiated ones (1.65 +/- 0.41 vs. 0.39 +/- 0.14, P < 0.05). CONCLUSION: RNA HOTAIR expressions were higher in ovarian cancer; it may play a role in ovarian cancer, and become a biomarker for malignant degree of ovarian cancer and may provide a novel therapeutic target for ovarian cancer.

[The use of human papillomavirus genotying kit based on gene chip technology for detecting and typing of human papillomavirus].

OBJECTIVE: To evaluate a new human papillomavirus (HPV) genotyping technique based on gene chip technology (HPG) for HPV genotyping and its clinical efficacy. METHODS: HPV genotyping (HPG) test, hybrid capture II (HC2) test and DNA sequencing assay were performed in 151 patients aged 20-75 years with diagnosis of chronic cervicitis or abnormal vaginal bleeding. The cervical specimens were collected from cervical epithelium. All the cervical samples were analyzed by the HPG test, HC2 test and DNA sequencing. The clinical efficacy of the HPG test was analyzed. RESULTS: The consistent rate between HPG test and HC2 test was 87.42% (kappa = 0.75, P < 0.05). When DNA sequencing assay was regarding as the final test result, the sensitivity and specificity of HPG test for high risk HPV were 100% and 96.49%, respectively. The consistent rate between HPG test and direct DNA sequencing was 98.70% (kappa = 0.97, P < 0.05). The most common six HPV genotypes detected by HPG test were HPV 16 (13.25%), 58 (11.92%), 52 (11.92%), 31 (6.62%) 39 (5.96%), 33 (5.96%) in descending order of frequency. The incidence of multiple-types infection detected by HPG test was 23.84%. CONCLUSION: HPG test is a rapid and accurate test for HPV genotyping which could detect 29 types of HPV infection at one time. It is suitable for cervical HPV infection screening in clinic.

Isoalantolactone mediates the degradation of BCR-ABL protein in imatinib-resistant CML cells by down-regulating survivin.

Isoalantolactone (Iso) is a bioactive lactone isolated from the root of Inula helenium L, which has been reported to have many pharmacological effects. To investigate the role and mechanism of isoalantolactone in chronic myeloid leukemia (CML), we first investigated isoalantolactone's anti-proliferative effects on imatinib-sensitive and imatinib-resistant CML cells by CCK8. Flow cytometry was used to detect isoalantolactone-induced cell apoptosis. Survivin was overexpressed in KBM5 and KBM5T315I cells using the lentivirus vector pSIN-3×flag-PURO. In KBM5 and KBM5T315I cells, shRNA was used to knockdown survivin. Cellular Thermal Shift Assay (CETSA) was used to detect the interaction between isoalantolactone and survivin. The ubiquitin of survivin induced by isoalantolactone was detected through immunoprecipitation. Quantitative polymerase-chain reaction (Q-PCR) and western blotting were used to detect the levels of mRNA and protein. Isoalantolactone inhibits the proliferation and promotes apoptosis of imatinib-resistant CML cells. Although isoalantolactone inhibits the proteins of BCR-ABL and survivin, it cannot inhibit survivin and BCR-ABL mRNA levels. Simultaneously, it was shown that isoalantolactone can degrade survivin protein by increasing ubiquitination. It was demonstrated that isoalantolactone-induced survivin mediated downregulation of BCR-ABL protein. It was also revealed that isoalantolactone triggered BCR-ABL protein degradation via caspase-3. Altogether, isoalantolactone inhibits survivin through the ubiquitin proteasome pathway, and mediates BCR-ABL downregulation in a caspase-3 dependent manner. These data suggest that isoalantolactone is a natural compound, which can be used as a potential drug to treat TKI-resistant CML.

[Observation on anticancer function of PS II isolated from Rhizoma Paridis].

OBJECTIVE: To isolate compound Paris saponin II (PS II) from Rhizoma Paridis and observe its antitumor activity. METHODS: PS II was isolated and determined by electrospray ionization-mass spectrometry, 1H and 13C nuclear magnetic resonance spectral analysis. PS II was used to treat cancer cells to analyze the toxicity and relative time-and dose-dependent manner. Apoptosis of cells were investigated by flow cytometry and TUNEL assay. Western blotting was used to analyze the effects of PS II to MAPKs and mitochondrial apoptotic pathway. RESULTS: The anti-tumor activities of PS II on ovarian carcinoma cell line SKOV3 were investigated. The IC50 of PS II to SKOV3 was 4.81 micromol/L. Increased apoptosis rate in a dose- and time-dependent manner was observed by Flow cytometry and TUNEL. The activity of ERK1/2 was inhibited by PS II and increased expression of cytochrone c, caspase-3 and caspase-9 was also noticed after the treatment of PS II . CONCLUSION: PS II can inhibit the growth of ovarian cancer cells SKOV3 through affecting the key proteins on MAPKs pathway and inhibiting the activity of ERK1/2 and eventually eliciting programmed cell death of SKOV3. The mitochondrial apoptotic pathway may be involved in the PSII induced apoptosis.

[Establishment of a cisplatin-resistant human cervical cancer cell line].

OBJECTIVE: To establish a cisplatin (cDDP)-resistant human cervical cancer cell line named SiHa/cDDP and researched its biological characteristics. METHODS: The development of cDDP resistance in SiHa cell line was induced by continuously stepwise exposure of the cells to cDDP. Cell growth curve, doubling time and resistance index (RI) were evaluated by MTT analysis. The expression of P-gp, GST-pi, Topo I were assessed with immunocytochemical method. RESULTS: SiHa/cDDP cell line was successfully established which had stable growth, subculture, frozen reservation and resuscitation in the concentration of 2 microg/ml cDDP, doubling time was (45.82 +/- 3.69) h and RI to cDDP was 16.131. It also showed different degrees of cross-resistance to several anticancer drugs. As compared to parental SiHa, SiHa/cDDP had over-expression in P-gp and GST-pi (P < 0.01), but Topo I showed no statistically significant differences. CONCLUSION: We successfully established the cDDP-resistant human cervical cancer cell line SiHa/cDDP, which may provide ideal experimental model for research of human cervical carcinoma.

[Dynamic detection of regulatory T cells in murine mammary carcinoma model and its significance].

OBJECTIVE: To analyze the dynamic change of regulatory T cells in experimental murine mammary carcinoma model, and to investigate their effect on tumor progress. METHODS: Mouse mammary carcinoma models were established. The percentages of CD4+ CD25+ and CD4+ FOXP3+ regulatory T cells in the CD4+ T cells in tumor tissue, tumor draining lymph node and spleen were measured by FACS at 3 different time points after tumor challenge. The expression of CD25+ FOXP3 in CD4+ T cells was analyzed, and the expression of CD4+ T cells in T cells (CD3+) of tumor tissue. RESULTS: Compared with the normal control group, In mouse mammary carcinoma models, The percentages of CD4+ T cells to lymphocytes in lymph node and spleen were decreased in the late stage (P<0.05). The percentages of CD4+ CD25+ and CD4+ FOXP3+ regulatory T cells in the CD4+ T cells were markedly increased (P<0.05), The percentages of CD25+ FOXP3+ regulatory T cells in the CD4+ T cells were markedly increased also (P<0.05). In the tumor tissue, the percentages of CD4+ T cells and CD4+ CD25+ regulatory T cells in T cells (CD3+) were increased in three weeks after the tumor challenge than one week (P< 0. 05). The percentages of CD4+ CD25+ and CD4+ FOXP3+ regulatory T cells in the CD4+ T cells have no change between one week and three weeks after tumor planting (P>0.05). CONCLUSION: Regulatory T cells were significantly increased in malignant tumor model, and closely related to the development of tumor.

[Isolation and biological characterization of human amniotic fluid-derived stem cells].

OBJECTIVE: To establish in vitro culture procedure for human amniotic fluid-derived CD117 positive stem cells, and to identify the characteristics of CD117 positive stem cells. METHODS: 86 amniotic fluid samples (10 mL of each) were obtained by second-trimester amniocentesis. Isolation of amniotic fluid-derived stem cells expressing CD117 antigen was performed via magnetic cell sorting using the CD117 MicroBead Kit. The karyotype of CD117 positive stem cells was analysed through repeated freezing. Adipogenic differentiation of these CD117 positive stem cells was displayed by Oil Red O staining. Osteogeneic differentiation of these CD117 positive stem cells was confirmed by Alizarin Red staining. RESULTS: The CD117 positive stem cells were successfully isolated and cultured from 61 samples, with all showing normal karyotype. Product analysis of specific staining confirmed that under specific culture mediums, these cells could be successfully induced to differentiate into adipocytes and osteocytes. CONCLUSION: Based on this study, we estimate that isolating CD117 positive stem cells from second-trimester amniotic fluid obtained by amniocentesis has a success rate of 70.93%. These cells maintain morphological and genetic stability in vitro. Human amniotic fluid-derived CD117 positive stem cells have the ability to differentiate in vitro into adipocytes and osteocytes under specific culture mediums and may be applied in cell transplantation and regenerative medicine.

[Protective effect of intralipid on myocardial ischemia/reperfusion injury in isolated rat heart].

OBJECTIVE: To investigate whether intralipid could protect perfused hearts of rats against ischemia/reperfusion (I/R) injury when it was administered before (preconditioning) or after the adverse ischemic event (postconditioning), in order to ascertain if intralipid would be a novel therapeutic strategy for myocardial I/R injury. METHODS: Studies were conducted in Langendorff-perfused isolated rat hearts. Twenty-four male Sprague-Dawley (SD) rats were divided into 3 groups randomly. The experimental procedure consisted of balance perfusion for 50 minutes, warm global ischemia (37 centigrade) for 40 minutes and 120 minutes of reperfusion. Preconditioning of hearts in myocardial preconditioning (I-preC group) consisted of 15 minutes of intralipid followed by 15 minutes of wash out before ischemia for 40 minutes and reperfusion for 120 minutes. In myocardial postconditioning (I-postC group) rat hearts were perfused with intralipid for 15 minutes at the onset of reperfusion. Hearts without intralipid treatment served as ischemic control (ISCH) group. Left ventricular mechanical function [heart rate (HR), left ventricular end-diastolic pressure (LVEDP), left ventricular diastolic pressure (LVDP), maximal change rate of intraventricular pressure rise/down (+/-dp/dt max)] were measured during the experiment, cardiac enzyme activity [creatine kinase (CK), lactate dehydrogenase (LDH)] were determined at 20 minutes after balance and 60 minutes after reperfusion. RESULTS: Comparing with ISCH group, the LVDP and +dp/dt max in the I-postC group were significantly higher and LVDEP, -dp/dt max were lower when compared with ISCH groups during reperfusion (all P<0.05). There were no significant differences in above indexes in I-preC group. As compared with the ISCH group, the content of LDH and CK in I-preC and I-postC were significantly lower at 60 minutes after reperfusion (all P<0.05). However, there were no significant differences between the I-preC and I-postC groups with respect to the levels of LDH and CK. The infarct size (IS) of I-preC and I-postC was markedly smaller than that of the ISCH group at 120 minutes of reperfusion [(21.6+/-1.8)%, (15.9+/-1.3)% vs. (46.5+/-3.9)%, both P<0.05]. The IS did not differ between the I-preC and I-postC groups (P>0.05). CONCLUSION: Intralipid administered before or after ischemia can decrease the level of CK, LDH and IS during reperfusion in isolated rat hearts. Intralipid postconditioning improves mechanical function.

[Protective effect of capsaicin on against myocardial ischemia-reperfusion injury of rat in vivo].

OBJECTIVE: To explore whether the capsaicin offers protective effect and possible mechanism on against myocardial ischemia-reperfusion injury of rat in vivo. METHODS: Ligating the left anterior descending coronary artery for 45 min and loosing it for 120 min were performed to establish the rat model of local myocardial ischemia-reperfusion injury in vivo. Forty healthy male rats were randomly divided into control group, capsaicin group, capsazepine group, and S-3144 group. In 10 min and 5 min before ischemia, all drugs for this study were delivered into rat left ventricle (LV) via right carotid artery. Variations of left ventricular function were successively monitored with ECG (electrocardiogram). Levels of creatine kinase MB (CKMB) in serum were determined. Evans-blue and TTC were utilized to identify the area at risk and the infarct size of LV. RESULTS: In all groups, the variables of left ventricular developed pressure (LVDP) significantly decreased after ischemia and reperfusion (P < 0.05). Except capsaicin group, heart rates (HR) of three other groups markedly reduced with reperfusion for 120 min as compared to pre-reperfusion (P < 0.05). In capsaicin group, there were significant increases in HR and LVDP with reperfusion for 120 min when compared with the other three groups (P < 0.05). At the end of experiment, the levels of CKMB and infract size were lower in capsaicin group than in the other three groups (P < 0.05), and there was no statistical significance among the other three groups (P > 0.05). CONCLUSION: Pretreatment with capsaicin can attenuate myocardial ischemia-reperfusion injury, of which the likely mechanism is by stimulating capsaicin receptor or TRPV1 and further activating substance P receptor in the rat in vivo.