[Circulating miR-152 helps early prediction of postoperative biochemical recurrence of prostate cancer].

OBJECTIVE: To investigate the value of circulating miR-152 in the early prediction of postoperative biochemical recurrence of prostate cancer. METHODS: Sixty-six cases of prostate cancer were included in this study, 35 with and 31 without biochemical recurrence within two years postoperatively, and another 31 healthy individuals were enrolled as normal controls. The relative expression levels of circulating miR-152 in the serum of the subjects were detected by qRT-PCR, its value in the early diagnosis of postoperative biochemical recurrence of prostate cancer was assessed by ROC curve analysis, and the correlation of its expression level with the clinicopathological parameters of the patients were analyzed. RESULTS: The expression of circulating miR-152 was significantly lower in the serum of the prostate cancer patients than in the normal controls (t = －5.212, P = 0.001), and so was it in the patients with than in those without postoperative biochemical recurrence (t = －5.727, P = 0.001). The ROC curve for the value of miR-152 in the early prediction of postoperative biochemical recurrence of prostate cancer showed the area under the curve (AUC) to be 0.906 (95% CI: 0.809－0.964), with a sensitivity of 91.4% and a specificity of 80.6%. The expression level of miR-152 was correlated with the Gleason score, clinical stage of prostate cancer, biochemical recurrence, and bone metastasis (P <0.05), decreasing with increased Gleason scores and elevated clinical stage of the malignancy. No correlation, however, was found between the miR-152 expression and the patients' age or preoperative PSA level (P >0.05). CONCLUSIONS: The expression level of circulating miR-152 is significantly reduced in prostate cancer patients with biochemical recurrence after prostatectomy and could be a biomarker in the early prediction of postoperative biochemical recurrence of the malignancy.

Clinical value of serum trophoblast cell surface protein 2 (TROP2) antibody in non-small-cell lung cancer patients.

OBJECTIVE: This study was to explore the clinical role of serum trophoblast cell surface protein 2 (TROP2) antibody in patients with non-small-cell lung cancer (NSCLC). MATERIALS AND METHODS: We collected serum specimens from 117 NSCLC patients, 40 benign lung disease patients, and 60 healthy controls. TROP2 antibody concentrations were measured using enzyme-linked immunosorbent assay. RESULTS: Serum TROP2 antibody levels were higher in the NSCLC group compared to the control group (p < 0.001). TROP2 antibody, at a cutoff value of 9.8 U/ml, showed good diagnostic performance for NSCLC. CONCLUSION: Measurement of TROP2 antibody might have diagnostic value for patients with NSCLC.

Mitochondrial COI/tRNASer(UCN) G7444A mutation may be associated with aminoglycoside-induced and non-syndromic hearing impairment.

Mutations in mitochondrial DNA (mtDNA) have been reported to have important roles in aminoglycoside-induced hearing impairment; however, the underlying molecular mechanisms have remained largely elusive. The current study presented a case of a Chinese patient with maternally inherited aminoglycoside-induced hearing impairment. A profound hearing impairment was identified by clinical evaluation; furthermore, analysis of the mitochondrial genome sequence of the patient revealed the presence of an A1555G mutation in the 12S rRNA as well as a G7444A mutation in the COI/tRNASer(UCN) gene. As the G7444A mutation is highly conserved between various species, it may be a modifying factor with regard to the pathological effects of the A1555G mutation.

Expression of ERCC1, MSH2 and PARP1 in non-small cell lung cancer and prognostic value in patients treated with platinum-based chemotherapy.

PURPOSE: To evaluate the prognostic value of the expression of excision repair cross-complementation group l (ERCC1), MutS protein homolog 2 (MSH2) and poly ADP-ribose polymerase 1 (PARP1) in non-small-cell lung cancer patients receiving platinum-based postoperative adjuvant chemotherapy. METHODS: Immunohistochemistry was applied to detect the expression of ERCC1, MSH2 and PARP1 in 111 cases of non-small cell lung cancer paraffin embedded surgical specimens. Through og-rank survival analysis, we evaluated the prognostic value of the ERCC1, MSH2, PARP1 and the related clinicopathological factors. COX regression analysis was used to determine whether ERCC1, MSH2 and PARP1 were independent prognostic factors. RESULTS: In the enrolled 111 non-small cell lung cancer patients, the positive expression rate of ERCC1, MSH2 and RARP1 was 33.3%, 36.9% and 55.9%, respectively. ERCC1 (P<0.001) and PARP1 (P=0.033) were found to be correlated with the survival time while there was no correlation for MSH2 (P=0.298). Patients with both ERCC1 and PARP1 negative cancer had significantly longer survival time than those with ERCC1 (P=0.042) or PARP1 (P=0.027) positive alone. Similalry, the survival time of patients with both ERCC1 and PARP1 positive cancer was shorter than those with ERCC1 (P=0.048) or PARP1 (P=0.01) positive alone. CONCLUSION: Patients with ERCC1 or PARP1 negative non-small cell lung cancer appear to benefit from platinum-based postoperative adjuvant chemotherapy.

Clinical value of eukaryotic elongation factor 2 (eEF2) in non-small cell lung cancer patients.

BACKGROUND: The purpose of this study was to evaluate a new type of tumor biomarker, eukaryotic elongation factor 2 (eEF2), in serum for the early diagnosis, confirmative diagnosis as well as assessment of treatment of non-small cell lung cancer (NSCLC). METHODS: 130 patients with NSCLC and 50 healthy individuals undergoing physical examination in our hospital provided the observation and healthy control groups. An enzyme linked immune sorbent assay (ELISA) method was applied to determine serum eEF2 levels. Serum neuron specific enolase (NSE) and squamous cell carcinoma antigen (SCC) levels in the observation group were assessed with an automatic biochemical analyzer. RESULTS: The median levels of eEF2 in the serum of NSCLC patients was found to be significantly higher than the healthy control group (p < 0.01) and it was markedly higher in stages III, IV than stages I, II (p < 0.05). eEF2 was higher with tumor size ≥ 2 cm than <2 cm (P< 0.01). Furthermore, two weeks after surgery patients showed a significant trend for eEF2 decrease (p < 0.05). CONCLUSIONS: The eukaryotic elongation factor 2 (eEF2) has certain clinical values for early diagnosis, verification, and prognosis as well as classification of lung cancer patients.

Regulation of the calcium-sensing receptor in both stomatal movement and photosynthetic electron transport is crucial for water use efficiency and drought tolerance in Arabidopsis.

Production per amount of water used (water use efficiency, WUE) is closely correlated with drought tolerance. Although stomatal aperture can regulate WUE, the underlying molecular mechanisms are still unclear. Previous reports revealed that stomatal closure was inhibited in the calcium-sensing receptor (CAS) antisense line of Arabidopsis (CASas). Here it is shown that decreased drought tolerance and WUE of CASas was associated with higher stomatal conductance due to improper regulation of stomatal aperture, rather than any change of stomatal density. CASas plants also had a lower CO2 assimilation rate that was attributed to a lower photosynthetic electron transport rate, leading to higher chlorophyll fluorescence. Gene co-expression combined with analyses of chlorophyll content and transcription levels of photosynthesis-related genes indicate that CAS is involved in the formation of the photosynthetic electron transport system. These data suggest that CAS regulates transpiration and optimizes photosynthesis by playing important roles in stomatal movement and formation of photosynthetic electron transport, thereby regulating WUE and drought tolerance.

Calcium-sensing receptor regulates stomatal closure through hydrogen peroxide and nitric oxide in response to extracellular calcium in Arabidopsis.

The Arabidopsis calcium-sensing receptor CAS is a crucial regulator of extracellular calcium-induced stomatal closure. Free cytosolic Ca(2+) (Ca(2+)(i)) increases in response to a high extracellular calcium (Ca(2+)(o)) level through a CAS signalling pathway and finally leads to stomatal closure. Multidisciplinary approaches including histochemical, pharmacological, fluorescent, electrochemical, and molecular biological methods were used to discuss the relationship of hydrogen peroxide (H(2)O(2)) and nitric oxide (NO) signalling in the CAS signalling pathway in guard cells in response to Ca(2+)(o). Here it is shown that Ca(2+)(o) could induce H(2)O(2) and NO production from guard cells but only H(2)O(2) from chloroplasts, leading to stomatal closure. In addition, the CASas mutant, the atrbohD/F double mutant, and the Atnoa1 mutant were all insensitive to Ca(2+)(o)-stimulated stomatal closure, as well as H(2)O(2) and NO elevation in the case of CASas. Furthermore, it was found that the antioxidant system might function as a mediator in Ca(2+)(o) and H(2)O(2) signalling in guard cells. The results suggest a hypothetical model whereby Ca(2+)(o) induces H(2)O(2) and NO accumulation in guard cells through the CAS signalling pathway, which further triggers Ca(2+)(i) transients and finally stomatal closure. The possible cross-talk of Ca(2+)(o) and abscisic acid signalling as well as the antioxidant system are discussed.

Calcium and calcium receptor CAS promote Arabidopsis thaliana de-etiolation.

As a second messenger, the free cytosolic calcium ion (Ca(2+)) plays important roles in many biochemical and physiological processes including photosynthesis in plants. In this study, we investigated morphological changes, chlorophyll accumulation and chloroplast development during early photomorphogenesis in etiolated seedlings of both Arabidopsis thaliana wild type (WT) and those with the antisense of CAS, a calcium sensor (CASas). Seedlings were grown at high, medium and low Ca(2+) concentrations to identify the roles of Ca(2+) and CAS in de-etiolation and chloroplast development. The results demonstrated that Ca(2+) and CAS are correlated with de-etiolation of A. thaliana after light exposure. High Ca(2+) significantly increased chlorophyll content and improved chloroplast development in both A. thaliana WT and CASas etiolated seedlings during de-etiolation. The analysis by western blot and real-time fluorescent quantitative polymerase chain reaction indicated that the expression levels of CAS mRNA and protein were upregulated by white light and external Ca(2+) significantly. Etiolated CASas plants showed much lower chlorophyll content and delay of chloroplast development as compared with WT plants, indicating that CAS functions in de-etiolation. All together, we concluded that the de-etiolation in A. thaliana was promoted by the high Ca(2+) concentration and CAS expression to a certain extent.

Emissions of nitric oxide from 79 plant species in response to simulated nitrogen deposition.

To assess the potential contribution of nitric oxide (NO) emission from the plants grown under the increasing nitrogen (N) deposition to atmospheric NO budget, the effects of simulated N deposition on NO emission and various leaf traits (e.g., specific leaf area, leaf N concentration, net photosynthetic rate, etc.) were investigated in 79 plant species classified by 13 plant functional groups. Simulated N deposition induced the significant increase of NO emission from most functional groups, especially from conifer, gymnosperm and C(3) herb. Moreover, the change rate of NO emission was significantly correlated with the change rate of various leaf traits. We conclude that the plants grown under atmospheric N deposition, especially in conifer, gymnosperm and C(3) herb, should be taken into account as an important biological source of NO and potentially contribute to atmospheric NO budget.

Hydrogen sulphide enhances photosynthesis through promoting chloroplast biogenesis, photosynthetic enzyme expression, and thiol redox modification in Spinacia oleracea seedlings.

Hydrogen sulphide (H(2)S) is emerging as a potential messenger molecule involved in modulation of physiological processes in animals and plants. In this report, the role of H(2)S in modulating photosynthesis of Spinacia oleracea seedlings was investigated. The main results are as follows. (i) NaHS, a donor of H(2)S, was found to increase the chlorophyll content in leaves. (ii) Seedlings treated with different concentrations of NaHS for 30 d exhibited a significant increase in seedling growth, soluble protein content, and photosynthesis in a dose-dependent manner, with 100 µM NaHS being the optimal concentration. (iii) The number of grana lamellae stacking into the functional chloroplasts was also markedly increased by treatment with the optimal NaHS concentration. (iv) The light saturation point (Lsp), maximum net photosynthetic rate (Pmax), carboxylation efficiency (CE), and maximal photochemical efficiency of photosystem II (F(v)/F(m)) reached their maximal values, whereas the light compensation point (Lcp) and dark respiration (Rd) decreased significantly under the optimal NaHS concentration. (v) The activity of ribulose-1,5-bisphosphate carboxylase (RuBISCO) and the protein expression of the RuBISCO large subunit (RuBISCO LSU) were also significantly enhanced by NaHS. (vi) The total thiol content, glutathione and cysteine levels, internal concentration of H(2)S, and O-acetylserine(thiol)lyase and L-cysteine desulphydrase activities were increased to some extent, suggesting that NaHS also induced the activity of thiol redox modification. (vii) Further studies using quantitative real-time PCR showed that the gene encoding the RuBISCO large subunit (RBCL), small subunit (RBCS), ferredoxin thioredoxin reductase (FTR), ferredoxin (FRX), thioredoxin m (TRX-m), thioredoxin f (TRX-f), NADP-malate dehydrogenase (NADP-MDH), and O-acetylserine(thiol)lyase (OAS) were up-regulated, but genes encoding serine acetyltransferase (SERAT), glycolate oxidase (GYX), and cytochrome oxidase (CCO) were down-regulated after exposure to the optimal concentration of H(2)S. These findings suggest that increases in RuBISCO activity and the function of thiol redox modification may underlie the amelioration of photosynthesis and that H(2)S plays an important role in plant photosynthesis regulation by modulating the expression of genes involved in photosynthesis and thiol redox modification.

Effects of calcium on seed germination, seedling growth and photosynthesis of six forest tree species under simulated acid rain.

We selected six tree species, Pinus massoniana Lamb., Cryptomeria fortunei Hooibr. ex Otto et Dietr., Cunninghamia lanceolata (Lamb.) Hook., Liquidambar formosana Hance, Pinus armandii Franch. and Castanopsis chinensis Hance, which are widely distributed as dominant species in the forest of southern China where acid deposition is becoming more and more serious in recent years. We investigated the effects and potential interactions between simulated acid rain (SiAR) and three calcium (Ca) levels on seed germination, radicle length, seedling growth, chlorophyll content, photosynthesis and Ca content in leaves of these six species. We found that the six species showed different responses to SiAR and different Ca levels. Pinus armandii and C. chinensis were very tolerant to SiAR, whereas the others were more sensitive. The results of significant SiAR × Ca interactions on different physiological parameters of the six species demonstrate that additional Ca had a dramatic rescue effect on the seed germination and seedling growth for the sensitive species under SiAR. Altogether, we conclude that the negative effects of SiAR on seed germination, seedling growth and photosynthesis of the four sensitive species could be ameliorated by Ca addition. In contrast, the physiological processes of the two tolerant species were much less affected by both SiAR and Ca treatments. This conclusion implies that the degree of forest decline caused by long-term acid deposition may be attributed not only to the sensitivity of tree species to acid deposition, but also to the Ca level in the soil.

Identification of cytokines involved in hepatic differentiation of mBM-MSCs under liver-injury conditions.

AIM: To identify the key cytokines involved in hepatic differentiation of mouse bone marrow mesenchymal stem cells (mBM-MSCs) under liver-injury conditions. METHODS: Abdominal injection of CCl(4) was adopted to duplicate a mouse acute liver injury model. Global gene expression analysis was performed to evaluate the potential genes involved in hepatic commitment under liver-injury conditions. The cytokines involved in hepatic differentiation of mBM-MSCs was functionally examined by depletion experiment using specific antibodies, followed by rescue experiment and direct inducing assay. The hepatic differentiation was characterized by the expression of hepatic lineage genes and proteins, as well as functional features. RESULTS: Cytokines potentially participating in hepatic fate commitment under liver-injury conditions were initially measured by microarray. Among the up-regulated genes determined, 18 cytokines known to closely relate to liver growth, repair and development, were selected for further identification. The fibroblast growth factor-4 (FGF-4), hepatocyte growth factor (HGF) and oncostatin M (OSM) were finally found to be involved in hepatic differentiation of mBM-MSCs under liver-injury conditions. Hepatic differentiation could be dramatically decreased after removing FGF-4, HGF and OSM from the liver-injury conditioned medium, and could be rescued by supplementing these cytokines. The FGF-4, HGF and OSM play different roles in the hepatic differentiation of mBM-MSCs, in which FGF-4 and HGF are essential for the initiation of hepatic differentiation, while OSM is critical for the maturation of hepatocytes. CONCLUSION: FGF-4, HGF and OSM are the key cytokines involved in the liver-injury conditioned medium for the hepatic differentiation of mBM-MSCs.

Recruitment of endogenous bone marrow mesenchymal stem cells towards injured liver.

Recent studies suggest that mesenchymal stem cells (MSCs) possess a greater differentiation potential than once thought and that they have the capacity to regenerate damaged tissues/organs. However, the evidence is insufficient, and the mechanism governing the recruitment and homing of MSCs to these injured sites is not well understood. We first examined the MSCs circulating in peripheral blood and then performed chemotaxis, wound healing and tubule-formation assays to investigate the migration capability of mouse bone marrow MSCs (mBM-MSCs) in response to liver-injury signals. In addition, BM-MSCs from donor enhanced green fluorescent protein transgenic male mice were transplanted into liver-injured co-isogenic female recipients, either by intra-bone marrow injection or through the caudal vein, to allow in vivo tracking analysis of the cell fate after transplantation. Donor-derived cells were analysed by in vivo imaging analysis, PCR, flow cytometry and frozen sections. Microarray and real-time PCR were used for chemokine/cytokine and receptor analyses. We successfully isolated circulating MSCs in peripheral blood of liver-injured mice and provided direct evidence that mBM-MSCs could be mobilized into the circulation and recruited into the liver after stimulation of liver injury. CCR9, CXCR4 and c-MET were essential for directing cellular migration towards the injured liver. The recruited mBM-MSCs may play different roles, including hepatic fate specification and down-regulation of the activity of hepatic stellate cells which inhibits over-accumulation of collagen and development of liver fibrosis. Our results provide new insights into liver repair involving endogenous BM-MSCs and add new information for consideration when developing clinical protocols involving the MSCs.

Direct hepatic differentiation of mouse embryonic stem cells induced by valproic acid and cytokines.

AIM: To develop a protocol for direct hepatic lineage differentiation from early developmental progenitors to a population of mature hepatocytes. METHODS: Hepatic progenitor cells and then mature hepatocytes from mouse embryonic stem (ES) cells were obtained in a sequential manner, induced by valproic acid (VPA) and cytokines (hepatocyte growth factor, epidermal growth factor and insulin). Morphological changes of the differentiated cells were examined by phase-contrast microscopy and electron microscopy. Reverse transcription polymerase chain reaction and immunocytochemical analyses were used to evaluate the gene expression profiles of the VPA-induced hepatic progenitors and the hepatic progenitor-derived hepatocytes. Glycogen storage, cytochrome P450 activity, transplantation assay, differentiation of bile duct-like structures and tumorigenic analyses were performed for the functional identification of the differentiated cells. Furthermore, FACS and electron microscopy were used for the analyses of cell cycle profile and apoptosis in VPA-induced hepatic differentiated cells. RESULTS: Based on the combination of VPA and cytokines, mouse ES cells differentiated into a uniform and homogeneous cell population of hepatic progenitor cells and then matured into functional hepatocytes. The progenitor population shared several characteristics with ES cells and hepatic stem/progenitor cells, and represented a novel progenitor cell between ES and hepatic oval cells in embryonic development. The differentiated hepatocytes from progenitor cells shared typical characteristics with mature hepatocytes, including the patterns of gene expression, immunological markers, in vitro hepatocyte functions and in vivo capacity to restore acute-damaged liver function. In addition, the differentiation of hepatic progenitor cells from ES cells was accompanied by significant cell cycle arrest and selective survival of differentiating cells towards hepatic lineages. CONCLUSION: Hepatic cells of different developmental stages from early progenitors to matured hepatocytes can be acquired in the appropriate order based on sequential induction with VPA and cytokines.

Induction of hepatic differentiation of mouse bone marrow stromal stem cells by the histone deacetylase inhibitor VPA.

Bone marrow stromal stem cells (BMSSCs) may have potential to differentiate in vitro and in vivo into hepatocytes. Here, we investigated the effects of valproic acid (VPA) involved in epigenetic modification, a direct inhibitor of histone deacetylase, on hepatic differentiation of mouse BMSSCs. Following the treatment of 2.5 mM VPA for 72 hrs, the in vitro expanded, highly purified and functionally active mouse BMSSCs from bone marrow were either exposed to some well-defined cytokines and growth factors in a sequential way (fibroblast growth factor-4 [FGF-4], followed by HGF, and HGF + OSM + ITS + dexamethasone, resembling the order of secretion during liver embryogenesis) or transplanted (caudal vein) in mice submitted to a protocol of chronic injury (chronic i.p. injection of CCl4). Additional exposure of the cells to VPA considerably improved the in vitro differentiation, as demonstrated by a more homogeneous cell population exhibited epithelial morphology, increasing expression of hepatic special genes and enhanced hepatic functions. Further more, in vivo results indicate that the pre-treatment of VPA significantly increased the homing efficiency of BMSSCs to the site of liver injury and, additionally, for supporting hepatic differentiation as well as in vitro. We have demonstrated the usefulness of VPA in the transdifferentiation of BMSSCs into hepatocytes both in vitro and in vivo, and regulation of fibroblast growth factor receptors (FGFRs) and c-Met gene expression through post-translational modification of core histones might be the primary initiating event for these effects. This mode could be helpful for liver engineering and clinical therapy.

Mesenchymal stem cells: a promising candidate in regenerative medicine.

Mesenchymal stem cells were initially characterized as plastic adherent, fibroblastoid cells. In recent years, there has been an increasing focus on mesenchymal stem cells since they have great plasticity and are potential for therapeutic applications. Mesenchymal stem cells or mesenchymal stem cell-like cells have been shown to reside within the connective tissues of most organs. These cells can differentiate into osteogenic, adipogenic and chondrogenic lineages under appropriate conditions. A number of reports have also indicated that these cells possess the capacity to trans-differentiate into epithelial cells and lineages derived from the neuro-ectoderm, and in addition, mesenchymal stem cells can migrate to the sites of injury, inflammation, and to tumors. These properties of mesenchymal stem cells make them promising candidates for use in regenerative medicine and may also serve as efficient delivery vehicles in site-specific therapy.

[Differentiation of adult mouse mesenchymal stem cells into hepatocytes cultured in a conditioned culture medium of injured hepatocytes].

OBJECTIVE: To establish a method through which murine bone marrow mesenchymal stem cells (MSCs) can be induced into hepatocytes in vitro. METHODS: A conditioned medium of injured hepatocytes (with CCl4 in vivo) was used to culture the isolated MSCs. The differentiated cells were identified by morphological observation, reverse transcription polymerase chain reaction (RT-PCR), immunofluorescence assay (for AFP, Albumin, and CK18) and periodic acid schiff reaction (PAS) for glycogen. RESULTS: The differentiated cells showed characteristics of hepatocytes. PT-PCR detected AFP mRNA on day 5 and it increased gradually until day 15, and then decreased; CK18 mRNA was detected on day 10; TAT was detected on day 20. Immunofluorescence assay for AFP, albumin and CK18 showed positive staining reactions on day 20. PAS positive glycogen granules appeared in the cytoplasm of the differentiated cells. CONCLUSION: MSCs of adult mice cultured in a conditioned medium of injured hepatocytes can differentiate into hepatocytes. This method can be used in further studying of the mechanism of transdifferentiation of MSCs into hepatocytes.

In vitro differentiation of mouse bone marrow stromal stem cells into hepatocytes induced by conditioned culture medium of hepatocytes.

The differentiation potential of adult stem cells has long been believed to be limited to the tissue or germ layer of their origin. However, recent studies have demonstrated that adult stem cells may encompass a greater potential than once thought. In the present study, we examined whether murine bone marrow derived stromal stem cells (BMSSCs) are able to differentiate into functional hepatocytes in vitro. BMSSCs were isolated from murine femora and tibiae, and the mesodermal multilineage differentiation potentials of these cells were functionally characterized. To effectively induce hepatic differentiation, we designed a novel protocol by using hepatocyte-conditioned medium. Hepatic differentiation from mouse BMSSCs was examined by a variety of assays at morphological and molecular levels. Morphologically, mouse BMSSCs became round and epithelioid, binucleated after induction. Differentiated cells were harvested on Days 0, 10, and 20 and subjected to examination of hepatocyte characteristics by reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry. We detected AFP, HNF-3beta, CK19, CK18, ALB, TAT, and G-6-Pase at the mRNA and/or protein levels, hepatocyte-like cells by culture in conditioned medium further demonstrated in vitro functions characteristic of liver cells, including glycogen storage, and urea secretion. Moreover, transplantation of the differentiated cells into liver-injured mice partially restored serum albumin level and significantly suppressed transaminase activity. Our findings indicated the transdifferentiation potential of mouse BMSSCs developing into the functional hepatocyte-like cells by conditioned culture medium and, hence, may serve as a model system for the study of mechanisms involved in the transdifferentiation, and a cell source for cell therapy of hepatic diseases.

[Establishment and application of fluorescent quantitative PCR method for detecting human herpes virus type 6].

OBJECTIVE: To establish fluorescent quantitative PCR method for detecting human herpes virus type 6 (HHV6). METHODS: According to the specific sequence of human herpes virus type 6 genes, the primers and the fluorescent probe (TaqMan) were designed and synthesized. The fragment generated from HHV 6 gene as template was cloned into the pMD18-T vector which was constructed from the pUC 18. And the positive recombinant plasmid was 1:10 diluted and used as standard quantitative template to make the standard curve for sample detection. RESULTS: The standard curve indicated the linear relationship between Ct (cycle threshold) and template concentration. The clinical samples from 135 cases were detected by this system, 16 cases among 135 were positive. CONCLUSION: The fluorescent quantitative PCR method for the detection of human herpes virus type 6 is simple and accurate, and this method may be helpful to clinical diagnosis.