A Golgi-Targeted Platinum Complex Plays a Dual Role in Autophagy Regulation for Highly Efficient Cancer Therapy.

Regulating autophagy to control the homeostatic recycling process of cancer cells is a promising anticancer strategy. Golgi apparatus is a substrate of autophagy but the Golgi-autophagy (Golgiphagy) mediated antitumor pathway is rarely reported. Herein, we have developed a novel Golgi-targeted platinum (II) complex Pt3, which is ca. 20 times more cytotoxic to lung carcinoma than cisplatin and can completely eliminate tumors after intratumoral administration in vivo. Its nano-encapsulated system for tail vein administration also features a good anti-tumor effect. Mechanism studies indicate that Pt3 induces substantial Golgi stress, indicated by the fragmentation of Golgi structure, down-regulation of Golgi proteins (GM130, GRASP65/55), loss of Golgi-dependent transport and glycosylation. This triggers Golgiphagy but blocks the subsequent fusion of autophagosomes with lysosomes, that is a dual role in autophagy regulation, resulting in loss of proteostasis and apoptotic cell death. As far as we know, Pt3 is the first Golgi-targeted Pt complex that can trigger Golgi stress-mediated dual-regulation of autophagic flux and autophagy-apoptosis crosstalk for highly efficient cancer therapy.

Mesenchymal stem cell-derived extracellular vesicles in skin wound healing: roles, opportunities and challenges.

Skin wounds are characterized by injury to the skin due to trauma, tearing, cuts, or contusions. As such injuries are common to all human groups, they may at times represent a serious socioeconomic burden. Currently, increasing numbers of studies have focused on the role of mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) in skin wound repair. As a cell-free therapy, MSC-derived EVs have shown significant application potential in the field of wound repair as a more stable and safer option than conventional cell therapy. Treatment based on MSC-derived EVs can significantly promote the repair of damaged substructures, including the regeneration of vessels, nerves, and hair follicles. In addition, MSC-derived EVs can inhibit scar formation by affecting angiogenesis-related and antifibrotic pathways in promoting macrophage polarization, wound angiogenesis, cell proliferation, and cell migration, and by inhibiting excessive extracellular matrix production. Additionally, these structures can serve as a scaffold for components used in wound repair, and they can be developed into bioengineered EVs to support trauma repair. Through the formulation of standardized culture, isolation, purification, and drug delivery strategies, exploration of the detailed mechanism of EVs will allow them to be used as clinical treatments for wound repair. In conclusion, MSC-derived EVs-based therapies have important application prospects in wound repair. Here we provide a comprehensive overview of their current status, application potential, and associated drawbacks.

Tertiary lymphoid structures and cytokines interconnections: The implication in cancer immunotherapy.

Tertiary lymphoid structures (TLSs) are organized aggregates of lymphocytes and antigen-presenting cells that develop in non-lymphoid tissues during chronic inflammation, resembling the structure and features of secondary lymphoid organs. Numerous studies have shown that TLSs may be an important source of antitumor immunity within solid tumors, facilitating T cell and B cell differentiation and the subsequent production of antitumor antibodies, which are beneficial for cancer prognosis and responses to immunotherapy. The formation of TLSs relies on the cytokine signaling network between heterogeneous cell populations, such as stromal cells, lymphocytes and cancer cells. The coordinated action of various cytokines drives the complex process of TLSs development. In this review, we will comprehensively describe the mechanisms by which various cytokines regulate TLS formation and function, and the recent advancements and therapeutic potential of exploiting these mechanisms to induce intratumoral TLSs as an emerging immunotherapeutic approach or to enhance existing immunotherapy.

Exosome-Mediated Immunosuppression in Tumor Microenvironments.

Exosomes are membranous structures secreted by nearly all cell types. As critical messengers for intercellular communication, exosomes deliver bioactive cargoes to recipient cells and are involved in multiple physiopathological processes, including immunoregulation. Our pioneering study revealed that cancer cells release programmed death-ligand 1-positive exosomes into the circulation to counter antitumor immunity systemically via T cells. Tumor cell-derived exosomes (TDEs) also play an immunosuppressive role in other immunocytes, including dendritic cells (DCs), macrophages, natural killer (NK) cells, and myeloid-derived suppressor cells (MDSCs). Moreover, exosomes secreted by nontumor cells in the tumor microenvironments (TMEs) also exert immunosuppressive effects. This review systematically provides a summary of the immunosuppression induced by exosomes in tumor microenvironments, which modulates tumor growth, invasion, metastasis, and immunotherapeutic resistance. Additionally, therapeutic strategies targeting the molecular mechanism of exosome-mediated tumor development, which may help overcome several obstacles, such as immune tolerance in oncotherapy, are also discussed. Detailed knowledge of the specific functions of exosomes in antitumor immunity may contribute to the development of innovative treatments.

Serum antiphospholipid antibody status may not be associated with the pregnancy outcomes of patients undergoing in vitro fertilization.

BACKGROUND: Antiphospholipid syndrome (APS) is an autoimmune disease that is associated with recurrent pregnancy loss. It is still controversial whether the presence of antiphospholipid antibodies (aPL) in the serum of patients with in vitro fertilization-embryo transfer (IVF-ET) has a negative effect on the outcomes. In view of the discrepancies, a meta-analysis of the published data was performed to explore the relationship of aPL and IVF-ET outcomes. METHODS: We searched for all published articles indexed in PubMed, Web of Science, and Cochrane Library, which were retrieved up to April, 2021. A total of 921 studies were yielded, of which 6 finally met the inclusion criteria. We carried out the meta-analysis by pooling results of these studies with Review Manager 5.3 software. The effect index was measured with 95% confidence intervals (CIs) of the relative risks (RRs). RESULTS: Six eligible studies were included in this meta-analysis, involving 3214 patients. Our results showed that positive aPL was not associated with decreased clinical pregnancy rate (RR 0.97; 95% CI 0.91-1.04). There was no correlation between positive aPL and increased miscarriage risk (RR 1.22; 95% CI 0.94-1.58). Only 5 of the 6 studies referred to live birth rate, but still no association was found between them (RR 0.95; 95% CI 0.81-1.11). CONCLUSIONS: The results showed that the presence of positive aPL neither decreased clinical pregnancy rate and live birth rate, nor increased miscarriage rate in women undergoing IVF, which is differed from the opinion of clinical practice. More prospective studies with high quality and larger sample size are needed to evaluate the relationship between positive aPL and outcomes of IVF-ET.

CNOT6/6L-mediated mRNA degradation in ovarian granulosa cells is a key mechanism of gonadotropin-triggered follicle development.

CCR4-NOT deadenylase is a major regulator of mRNA turnover. It contains two heterogeneous catalytic subunits CNOT7/8 and CNOT6/6L in vertebrates. The physiological function of each catalytic subunit is unclear due to the gene redundancy. In this study, Cnot6/6l double knockout mice are generated. Cnot6l-/- female mice are infertile, with poor ovarian responses to gonadotropins. Follicle-stimulating hormone (FSH) stimulates the transcription and translation of Cnot6 and Cnot6l in ovarian granulosa cells. CNOT6/6L function as key effectors of FSH in granulosa cells and trigger the clearance of specific transcripts in granulosa cells during preantral to antral follicle transition. These results demonstrate that FSH modulates granulosa cell function by stimulating selective translational activation and degradation of existing mRNAs, in addition to inducing de novo gene transcription. Meanwhile, this study provides in vivo evidence that CNOT6/6L-mediated mRNA deadenylation is dispensable in most somatic cell types, but is essential for female reproductive endocrine regulation.

A positive COX-2/IL-1ß loop promotes decidualization by upregulating CD82.

A successful pregnancy requires sufficient decidualization of endometrial stromal cells (ESCs). CD82, a metastasis suppressor, is a critical regulator for trophoblast invasion but the effect in decidualization was largely unknown. Here we reported that there was a high level of CD82 in DSC by the immunohistochemistry staining and flow cytometer analysis. Stimulation with prostaglandin E2 (PGE2) elevated the expression of CD82 in ESCs. In contrast, celecoxib, a selective COX-2 inhibitor, significantly downregulated the expression of CD82 in decidual stromal cells (DSCs). Bioinformatics analysis and further research showed that recombinant human interleukin (IL)-1ß protein (rhIL-1ß) upregulated CD82 in ESCs. Of note, blocking IL-1ß signaling with anti-human IL-1ß neutralizing antibody could reverse the stimulatory effect of PGE2 on CD82 in ESCs. Silencing CD82 resulted in the decease of the decidualization markers PRL and IGFBP1 mRNA levels in DSCs. More importantly, we observed rhIL-1ß also upregulated the expression of COX-2, and the upregulation of PRL and IGFBP1 induced by rhIL-1ß could be abolished by celecoxib in ESCs or CD82 deficiency in DSCs. This study suggests that CD82 should be a novel promotor for decidualization under a positive regulation of the COX-2/PGE2/IL-1ß positive feedback loop.

[Deletion of extracellular loops of occludin affects the tight junction of TM4 cells in mice].

OBJECTIVE: To analyze the functions of the two extracellular loops of occludin in the tight junction of TM4 cells in mice. METHODS: Using genetic engineering, we separately or simultaneously deleted two extracellular loops of occludin, cloned the three occludin genes without extracellular loops into the pcDNA3.1 expression vector, and transfected them into TM4 cells. Then we determined the expression of occludin by RT-PCR and Western blot, and analyze the effects of the extracellular loops of occludin on the tight junction of the TM4 cells with the in vitro cell line model. RESULTS: The results of sequencing showed that the expression vector of pcDNA3.1 - occludin Δ OCC1, pcDNA3.1 - occludin Δ OCC2 and pcDNA3.1 - occludin Δ OCC1 + OCC2 was constructed successfully. The mRNA and protein expressions of occludin in the non-extracellular loop groups were significantly higher than in the control group. Both the extracellular loops of occludin increased the tight junction of the TM4 cells. The macromolecular permeability in the TM4 cells was significantly lower in the pcDNA3.1 - occludin Δ OCC1 than in the pcDNA3.1 - occludin Δ OCC2 group (P < 0.05), indicating a higher impact of the second than the first extracellular loop on the tight junction of the TM4 cells. CONCLUSIONS: Both of the two extracellular loops of occludin can affect the tight junction of TM4 cells, the second even more significantly than the first one.

Effect of interventional embolization treatment for hydrosalpinx on the outcome of in vitro fertilization and embryo transfer.

To explore the effect of interventional embolization treatment for hydrosalpinx on the outcome of in vitro fertilization and embryo transfer (IVF-ET).During the period from January 2013 to January 2015, a total of 129 patients with unilateral or bilateral hydrosalpinx were treated with IVF-ET and selected for retrospective analysis. Seventy-three patients (intervention group) with unilateral or bilateral hydrosalpinx were treated with fallopian tube embolization, which was followed by IVF-ET. During the same period, 56 patients (control group) with unilateral or bilateral hydrosalpinx directly received IVF-ET without receiving any treatment for hydrosalpinx.The clinical pregnancy rate of the control group was significantly lower than that of the intervention group (P < .05), while the abortion rate and ectopic pregnancy rate of the control group were strikingly higher than that of the intervention group (P < .05).Hydrosalpinx can decrease the clinical pregnancy rate of IVF-ET, and increase the incidence of abortion and ectopic pregnancy. The interventional embolization treatment for hydrosalpinx before IVF-ET can improve the clinical pregnancy rate and reduce adverse pregnancy outcome and which, with the advantages of a high success rate, convenient use, low cost, less pain, no anesthetic risk and no effect on the ovarian function it may further be developed for use in the clinic.

[Deletion of the env region in HERV-H-X gene and its expression in colon cancer].

BACKGROUND & OBJECTIVE: We have identified a novel gene HERV-H-X from colon cancer tissue recently. This research was to analyze the deletion of the env region of HERV-H-X, to compare its expression with that of the env open reading frames (ORFs) in other HERV-H proviruses, and to study its differential expression in colon cancer and normal colon tissues. METHODS: Multiple alignments were performed for HERV-H-X and 3 other HERV-H proviruses (HERV-H/env62, HERV-H/env60, and HERV-H/env59) containing intact env ORF. HERV-H-X-specific polymerase chain reaction (PCR) and env ORF-common PCR for HERV-H/env62, env60 and env59 were conducted to study their expression in 8 pairs of colon cancer and adjacent normal tissues. Real-time quantitative PCR was conducted to study the differential expression of HERV-H-X in 17 pairs of colon cancer and adjacent normal tissues. RESULTS: The missing part in the env region of HERV-H-X corresponded to env ORFs in HERV-H/env62, HERV-H/env60 and HERV-H/env59. HERV-H-X was only detected in colon cancer tissues, while env ORFs were detected both in cancer and normal tissues. The expression of HERV-H-X was up-regulated in colon cancer tissues by 24.9 folds of that in normal tissues (P<0.01). CONCLUSION: HERV-H-X is highly expressed in colon cancer, but its up-regulation is not related to env gene as it lacks an env ORF.

[Growth inhibition of MG-63 cells by cyclin A2 gene-specific small interfering RNA].

OBJECTIVE: To study the impact of small interference RNA (siRNA) targeting cyclin A2 gene on the growth of MG-63 and HSF cells and to explore whether cyclin A2 siRNAs could become a useful tool in the treatment of osteosarcoma. METHODS: One pair of siRNA targeting the cyclin A2 mRNA and a pair of nonsense siRNA were designed according the current criteria. SiRNA was chemically synthesized and purified. The siRNA was transfected into osteosarcoma cell line MG-63 and normal human skin fibroblast (HSF) cells via oligofectamine. Cells transfected with nonsense siRNA served as the negative control and those only treated with PBS as the blank control group. Quantitative fluorescence RT-PCR, Western-blot, MTT assay, reverse transcriptase (RT)-PCR, flow cytometry and colony-forming test were employed to evaluate the efficacy of RNA interference. At the same time, the mRNA expression of PCNA and cyclin B1 in siRNA treated MG-63 cells were examined. RESULTS: 1 nmol/L, 10 nmol/L, 50 nmol/L and 100 nmol/L cyclin A2-siRNA can reduced cyclin A2 mRNA and protein expression respectively by 9.43%, 56.35%, 79.17% and 84.30% as compared with that of the blank control group, whereas the negative and blank control groups had similar expression levels. After 48 h treatment with 10 nmol/L siRNA, MG-63 cells were arrested in G0/G1 phase and the proliferation of this tumor cell was suppressed by 39.06% 48 h after transfection. Furthermore, the treated MG-63 cells showed less colony-forming ability. Increasing the siRNA concentration to 50 nmol/L can further inhibit the proliferation of MG-63 cells by 54.94%. In addition, the cyclin A2-depleted MG-63 cells showed decreased levels of PCNA and cyclin B1. In contrast, although cyclin A2 mRNA and protein expression in HSF reduced 58.13% 48 h after treatment by 50 nmol/L siRNA, these cells exhibited only a slight change in cell cycle, and no clear inhibition of proliferation and impaired plate colony-forming ability was observed. CONCLUSION: Cyclin A2 gene maybe served as a potential target for tumor therapy. RNA interference induces obvious inhibition of cyclin A2 mRNA and protein expression in MG-63 and HSF cells, which consequently downregulate the proliferation of MG-63 cells. There is few inhibitory effect on the proliferation by siRNAs for HSF cells. These results indicate that siRNAs against cyclin A2 could become a potential antiproliferative tool in future antitumor therapy.

Discrimination of Lonicera japonica THUNB. from different geographical origins using restriction fragment length polymorphism analysis.

Lonicera japonica THUNB. is a commonly used anti-inflammatory herbal medicine. The therapeutic effectiveness of L. japonica depends significantly on its geographical origin. However, it is difficult to define criteria for confirming geographical authenticity using microscopic and chemical characteristics. In the present study, the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA loci of L. japonica from different origins and related species was sequenced. The mutation site found in the ITS region from geo-authentic L. japonica can be recognized by the restriction endonuclease EcoN I. Since PCR products from geo-authentic L. japonica cannot be digested completely, a quantitative restriction fragment length polymorphism analysis method was developed. The cleavage rate of PCR products by EcoN I was determined to be more than 70% in all geo-authentic L. japonica and less than 20% in non-geo-authentic L. japonica and other species from genus Lonicera. The rate correlated remarkably with the geographical origin of L. japonica. Therefore, this method can be used to classify geo-authentic L. japonica.

[Knockdown of cyclin A2 expression by small interfering RNA in MG-63 cells].

OBJECTIVE: To study the inhibitory effect of small interference RNA (siRNA) targeting cyclin A2 gene on the growth of osteosarcoma MG-63 and human normal skin fibroblast HSF cells and to explore whether cyclin A2 siRNAs could become a useful tool in the treatment of osteosarcoma. METHODS: Three pairs of siRNAs targeting cyclin A2 mRNA and a pair of nonsense siRNA were designed according to the current criteria. SiRNAs were chemically synthesized and purified. The siRNAs were transfected into MG-63 cells and HSF cells via oligofectamine. The cells transfected with nonsense siRNA served as negative control group and those only treated with PBS as blank control group. Quantitative fluorescence RT-PCR, Western-blot, MTT assay, reverse transcriptase (RT)-PCR, flow cytometry and clone forming test were employed to evaluate the efficacy of RNA interference. At the same time, the mRNA expression of PCNA and cyclin B1 in siRNA-treated MG-63 cells were examined. RESULTS: Although all three siRNAs could reduce the cyclin A2 expression, siRNA, appeared to be the most effective. After 48 h treatment with siRNA1, cyclin A2 mRNA and protein expression in MG-63 cells was significantly reduced by nearly 80% as compared with that of the blank control group, whereas the negative and blank control groups had similar expression levels. MG-63 cells treated with siRNA1 were arrested at G0/G1 phase by 80.1% and the proliferation of these tumor cells was suppressed 48 h after transfection. Furthermore, MG-63 cells showed a decreased colony forming ability after siRNA1 treatment. In addition, the cyclin A2-depleted MG-63 cells showed decreased levels of PCNA and cyclin B1. In contrast, although cyclin A2 expression in HSF reduced by nearly 60% after treatment by siRNA1 for 48h, these cells exhibited only a slight change in cell cycling, and neither clear inhibition of proliferation nor impaired colony forming ability was observed. CONCLUSION: Cyclin A2 is critical for proliferation of MG-63 cells. Cyclin A2-siRNAs can induce obvious inhibition of cyclin A2 mRNA and protein expression in MG-63 and HSF cells, which consequently down-regulate the proliferation of MG-63 cells. There is little effect on the proliferation of siRNA-treated HSF cells. Those results indicate that siRNAs against cyclin A2 may become a potential antiproliferative tool in future antitumor therapy.

[Knockdown of PLK1 mRNA by special siRNA].

To study knockdown effect of small interfering RNA (siRNA) to PLK1 (Polo-like kinase 1) mRNA in colorectal cancer cell line SW480 and its mitosis and growth was changed. Ten special siRNA molecules were designed targeting different sites of PLK1 mRNA sequence and chemically synthesized. The siRNA molecules were transfected into SW480 by Oligofectamine. The gene mRNA level was assayed by Real-Time PCR. The changes of PLK1 protein, SW480 cell cycle and survival percentage was checked by Western-blot, Flow cytometry and Cell counter assays respectively. All 10 siRNA molecules knocked PLK1 mRNA down in different level. Of them P1, P4 and P9 showed over 80% knockdown efficiency and the others had more than 20% knockdown efficiency to PLK1 mRNA. The best knockdown effect over 95% of all groups was at 25 nmol/L of a mixture with P1, P4 and P9 siRNA equally. In this situation the protein was very less and the cells were blocked at G2 phase of cell cycle. After 72 h cell survival percentages were consistent with PKL1 mRNA level change by siRNA gradient concentration. The results showed that siRNA targeting PLK1 mRNA had effectively knocked PLK1 mRNA down in SW480 cell line. And a blended siRNAs held the best knockdown effect. The cell was blocked on the mitosis and growth.

Evaluation of ST13 gene expression in colorectal cancer patients.

We identified a novel gene ST13 from a subtractive cDNA library of normal intestinal mucosa in 1993, more studies showed that ST13 was a co-chaperone of Hsp70s. Recently we detected the ST13 gene expression in tumor tissue and adjacent normal tissue of the same colorectal cancer patient and investigated if the ST13 gene expression might have any prognostic value. Analysis was performed at molecular level by reverse transcription-PCR using real-time detection method. We measured two genes simultaneously, ST13 as the target gene and glyceraldehydes-3-phosphate dehydrogenase as a reference gene, in primary colorectal tumor specimens and tumor-adjacent normal mucosa specimens from 50 colorectal cancer patients. The expression levels of the ST13 gene were significantly decreased in primary tumors compared with adjacent mucosa (P<0.05). But there were no significant differences in the expression of ST13 as compared with different Dukes' stage, tumor differentiation grade, invasion depth, lymph node metastasis and disease-specific survival.

[Inhibition of hepatitis B virus by combined siRNAs in vitro].

Small interfering RNA (siRNA) technology is a powerful tool to knockdown gene expression in mammalian cells including genes of viral origin. To study the inhibition of hepatitis B virus (HBV) by combined siRNAs in vitro, HepG2 2.2.15, a human hepatoblastoma cell that constitutively produces HBV particles, was transfected with 12 siRNAs that were designed to target different regions on HBV genome. Enzyme-linked immunosorbent assay was used to measure the HBsAg and HBeAg levels in culture media, and HBV replication was determined by real-time quantitative PCR. The results showed that HBV replication could be inhibited by treatment with these siRNAs in a dose-dependent manner. The expression of HBsAg was inhibited by 80% and 90% with 80 nmol/L single siRNA treatment and 20 nmol/L combined siRNAs treatment, respectively. HBeAg was inhibited by about 60% with single siRNA treatment, and the inhibition didn't increase much in the case of combined siRNAs treatment. HBV replication was inhibited by 90% with 80 nmol/L single siRNA treatment or 20 nmol/L combined siRNAs treatment. These data provide solid evidence for developing new approaches in HBV treatment with combined siRNAs.

Apoptosis induction with polo-like kinase-1 antisense phosphorothioate oligodeoxynucleotide of colon cancer cell line SW480.

AIM: To investigate the effects of polo-like kinase-1 (PLK1) antisense phosphorothioate oligodeoxynucleotide (ASODN) on apoptosis and cell cycle of human colon cancer cell line SW480. METHODS: After SW480 colon cancer cells were transfected with PLK1 ASODN, Northern and Western blot analyses were used to examine PLK1 gene expression in cancer cells. We studied apoptosis using terminal uridine deoxynucleotidyl nick end labeling. Apoptosis and cell cycle of SW480 cells were examined by fluorescence-activated cell sorter scan. RESULTS: The levels of PLK1 mRNA and protein were greatly inhibited by PLK1 ASODN in SW480 cancer cells transfected with PLK1 ASODN. Apoptosis index (AI) induced PLK1 ASODN in a time- and dose-dependent manner. Results from FLM showed that sub-2N DNA content of transfected cancer cells was significantly increased and arrested at G2/M compared with control groups. CONCLUSION: PLK1 ASODN can induce apoptosis of human colon cancer cell line SW480.

Identification of Fritillaria pallidiflora using diagnostic PCR and PCR-RFLP based on nuclear ribosomal DNA internal transcribed spacer sequences.

Fritillaria pallidiflora Schrenk (Liliaceae) is a commonly used antitussive herb. There are 9 species of Fritillaria recorded as herbal drugs in the Chinese Pharmacopoeia. The other species are often marketed as F. pallidiflora, and thus, the therapeutic effects of F. pallidiflora are not achieved. Methods to distinguish F. pallidiflora from the 8 other species of Fritillaria are limited by the current morphological and chemical methods. In this study, we report two molecular authentication methods based on the sequences of nuclear ribosomal DNA internal transcribed spacer (nrDNA ITS) regions. For diagnostic PCR, we designed a pair of species-specific primers to authenticate F. pallidiflora. The PCR program consisted of only two steps for every repeated cycle. For PCR-RFLP, we identified a distinctive site which can be recognized by the restriction endonuclease Eco81I in the nrDNA ITS1 region of F. pallidiflora. PCR-RFLP analysis was established to differentiate F. pallidiflora from the other species of Fritillaria. These methods provide effective and accurate identification of F. pallidiflora.

Combination of small interfering RNAs mediates greater inhibition of human hepatitis B virus replication and antigen expression.

OBJECTIVES: To evaluate the inhibitory effect mediated by combination of small interfering RNAs (siRNAs) targeting different sites of hepatitis B virus (HBV) transcripts on the viral replication and antigen expression in vitro. METHODS: (1) Seven siRNAs targeting surface (S), polymerase (P) or precore (PreC) region of HBV genome were designed and chemically synthesized. (2) HBV-producing HepG2.2.15 cells were treated with or without siRNAs for 72 h. (3) HBsAg and HBeAg in the cell culture medium were detected by enzyme-linked immunoadsorbent assay. (4) Intracellular viral DNA was quantified by real-time PCR (Polymerase Chain Reaction). (5) HBV viral mRNA was reverse transcribed and quantified by real-time PCR. (6) The change of cell cycle and apoptosis was determined by flow cytometry. RESULTS: Our data demonstrated that synthetic small interfering RNAs (siRNAs) targeting S and PreC gene could efficiently and specifically inhibit HBV replication and antigen expression. The expression of HBsAg and HBeAg and the replication of HBV could be specifically inhibited in a dose-dependent manner by siRNAs. Furthermore, our results showed that the combination of siRNAs targeting various regions could inhibit HBV replication and antigen expression in a more efficient way than the use of single siRNA at the same final concentration. No apoptotic change was observed in the cell after siRNA treatment. CONCLUSION: Our results demonstrated that siRNAs exerted robust and specific inhibition on HBV replication and antigen expression in a cell culture system and combination of siRNAs targeting different regions exhibited more potency.

[Studies on influence of fungal elicitor on hairy root of Panax ginseng biosynthesis ginseng saponin and biomass].

OBJECTIVE: To study influence of fungal elicitors on the biomass and ginseng saponin biosynthesis of hairy roots of Panax ginseng (HRPG). METHOD: Fungal elicitors were extracted from Colletorichum lagnarinm, Phoma filtrate, Fusarium oxysponum, Asperillus niger and culture with HRPG. The total ginseng sponin and four kinds of monomeric sponins were analysed by UV-spectrophotometry and RP-HPLC. RESULT: Fungal elicitors coula not only can influence on HRPG biomass and total ginseng sponin, but also improve or decrease some monomeric sponin. The total ginseng sponin could be increased to 3.649% but Rg1 and Re could not be detected when A. niger elicitors wss 20 mg x L(-1) in the culture fluid. CONCLUSION: Fungal elicitor has specificity influence on secondary metabolite of HRPG. HRPG can biosynthesize specially active component by using specific fungal elicitor is used.