Ginsenoside Rb1, Compound K and 20(S)-Protopanaxadiol Attenuate High-Fat Diet-Induced Hyperlipidemia in Rats via Modulation of Gut Microbiota and Bile Acid Metabolism.

Hyperlipidemia, characterized by elevated serum lipid concentrations resulting from lipid metabolism dysfunction, represents a prevalent global health concern. Ginsenoside Rb1, compound K (CK), and 20(S)-protopanaxadiol (PPD), bioactive constituents derived from Panax ginseng, have shown promise in mitigating lipid metabolism disorders. However, the comparative efficacy and underlying mechanisms of these compounds in hyperlipidemia prevention remain inadequately explored. This study investigates the impact of ginsenoside Rb1, CK, and PPD supplementation on hyperlipidemia in rats induced by a high-fat diet. Our findings demonstrate that ginsenoside Rb1 significantly decreased body weight and body weight gain, ameliorated hepatic steatosis, and improved dyslipidemia in HFD-fed rats, outperforming CK and PPD. Moreover, ginsenoside Rb1, CK, and PPD distinctly modified gut microbiota composition and function. Ginsenoside Rb1 increased the relative abundance of Blautia and Eubacterium, while PPD elevated Akkermansia levels. Both CK and PPD increased Prevotella and Bacteroides, whereas Clostridium-sensu-stricto and Lactobacillus were reduced following treatment with all three compounds. Notably, only ginsenoside Rb1 enhanced lipid metabolism by modulating the PPARγ/ACC/FAS signaling pathway and promoting fatty acid ß-oxidation. Additionally, all three ginsenosides markedly improved bile acid enterohepatic circulation via the FXR/CYP7A1 pathway, reducing hepatic and serum total bile acids and modulating bile acid pool composition by decreasing primary/unconjugated bile acids (CA, CDCA, and ß-MCA) and increasing conjugated bile acids (TCDCA, GCDCA, GDCA, and TUDCA), correlated with gut microbiota changes. In conclusion, our results suggest that ginsenoside Rb1, CK, and PPD supplementation offer promising prebiotic interventions for managing HFD-induced hyperlipidemia in rats, with ginsenoside Rb1 demonstrating superior efficacy.

Improving the ability of CAR-T cells to hit solid tumors: Challenges and strategies.

Chimeric antigen receptor T cell (CAR-T) therapy is a late-model of immune cell therapy that has been shown to be effective in refractory/recurrent B-cell leukemia and lymphoma. Compared with the traditional anti-tumor methods, CAR-T cell therapy has the advantages of higher specificity, stronger lethality and longer-lasting efficacy. Although CAR-T cells have made significant progress in the treatment of hematologic malignancies, diverse difficulties remain in the treatment of solid tumors, including immune escape due to tumor antigen heterogeneity, preventing entry or limiting the persistence of CAR-T cells by physical or cytokine barriers and along with other immunosuppressive molecule and cells in the tumor microenvironment (TME). Otherwise, the intracellular signaling of CAR also impact on CAR-T cells persistence. Appropriate modification of intracellular costimulatory molecular signal in the structure of CAR or coexpression of CAR and cytokines can provide a way to enhance CAR-T cells activity. Additionally, CAR-T cells dysfunction due to T cell exhaustion is associated with multi-factors, especially transcription factors, such as c-Jun, NR4A. Engineering CAR-T cells to coexpress or knockout transcription factors in favor of TCM memory CAR-T cells differentiation was proved to prolonged the survival of CAR-T cells. Finally, combination of CAR-T cells with oncolytic viruses, nanoparticles or immune checkpoint inhibitors provides an effective measure to improve CAR-T cells function. Here, we discuss all of these advances and challenges and review promising strategies for treating solid tumors. In particular, we also highlight that CAR-T cells have enormous potential to be used in combination with other immunotherapies.

The protective effects of chronic intermittent hypobaric hypoxia pretreatment against collagen-induced arthritis in rats.

OBJECTIVE: To explore the immunological mechanisms underlying the effect of chronic intermittent hypobaric hypoxia (CIHH) pretreatment on collagen-induced arthritis (CIA) in rat. METHODS: Fifty-four adult male Sprague-Dawley rats were used in the experiment. Arthritis in CIA rats (n=18) was induced by injection of collagen. The CIHH+CIA rats (n=18) were treated with CIHH (simulated 3000 m altitude, 5 hours per day for 28 days, PO2=108.8 mmHg) before CIA. The control rats (n=18) were not given any treatment. RESULTS: (1) Incidence rate of CIA in CIHH+CIA rats was significantly lower than that in CIA rats (P<0.05). (2) The paw thickness and arthritis index (AI) value in CIHH+CIA rats were lower than those in CIA rats (P<0.05). (3) The hyperplasia with inflammatory infiltration in synovial tissue of joints in CIHH+CIA rats was much alleviative compared with CIA rats. (4) TNF-α, IFN-γ, IL-4 and IL-17 in synovial tissue of joint and serum in CIHH+CIA rats were decreased compared with CIA rats (P<0.05). (5) The number of CD4-positive T-lymphocytes and the ratio of CD4/CD8 T-lymphocytes in peripheral blood in CIHH+CIA rats were lower than those in CIA rats (P<0.05). (6) The protein expression of HIF-1α and NF-κB in synovial tissue of joint in CIHH+CIA rats was decreased compared with CIA rats (P<0.05). CONCLUSION: CIHH pretreatment has a protective effect against collagen-induced arthritis in rat through down-regulation of HIF-1α and NF-κB, inhibition of inflammatory cytokines TNF-α and IL-17, and balance in CD4/CD8 and Th1/Th2 T lymphocytes.

Plasminogen activator inhibitor-1 promotes the proliferation and inhibits the apoptosis of pulmonary fibroblasts by Ca(2+) signaling.

AIM: Our previous investigation demonstrated that plasminogen activator inhibitor-1 (PAI-1) siRNA ameliorated bleomycin (BLM)-induced rat lung fibrosis. The present study was undertaken to explore the effect and the mechanism of PAI-1 siRNA and plasmid pcDNA on the proliferation and apoptosis of cultured fibroblasts from BLM-induced fibrotic lung tissues. MATERIALS AND METHODS: The fibroblasts from BLM-induced fibrotic lung tissue were isolated and transfected using PAI-1 siRNA and plasmid pcDNA-PAI-1. The techniques of real time RT-PCR and/or western blot were used to determine the expression of PAI-1, α-smooth muscle actin (α-SMA) (real time RT-PCR only), collagen type-1 and type-3 (real time RT-PCR only), and the levels of caspase-3, ERK and AKT signal molecules. The proliferation of fibroblasts was measured by cell cycle with flow cytometry. The intracellular concentration of Ca(2+) was examined by confocal laser microscopy. RESULTS: PAI-1 siRNA downregulated the PAI-1 mRNA expression by 70%±7% at 24h and protein expression by 73.5%±10% and 42%±3% at 48h and 72h compared to Non-specific siRNA group. Flow cytometry showed that the fibroblasts at the G(2)M+S phase were significantly reduced by 20.56±1.03% after transfecting PAI-1 siRNA and were significantly increased by 43.8±1.21% after transfecting plasmid pcDNA-PAI-1. The mRNA expressions of α-SMA, collagen type-1and type-3 were downregulated after transfecting the PAI-1 siRNA, while upregulated after the transfection of pcDNA-PAI-1. PAI-1 siRNA increased the level of caspase-3, inhibited the expressions of p-ERK and p-AKT protein molecules, while the pcDNA-PAI-1 transfection showed a reversal effect on these expressions. Intracellular Ca(2+) concentration was decreased after transfecting PAI-1 siRNA, whereas increased after transfecting pcDNA-PAI-1. CONCLUSION: PAI-1 promotes the proliferation, transforming into myofibroblasts, collagen synthesis, and inhibits apoptosis of pulmonary fibroblasts by activating Ca(2+), ERK and AKT signaling pathway. Decreasing PAI-1 expression is an available strategy in inhibiting the progression of pulmonary fibrosis.

[Anti-tumor effect of cisplatin combined with DC vaccine on tumor-bearing mice].

OBJECTIVE: To explore the anti-tumor mechanism of the combination of cisplatin with DC vaccine in tumor-bearing mice. METHODS: B16 melanoma cells were treated with cisplatin at the final concentration of 20 µg/ml in vitro for 24 h. The expression of HMGB1, Hsp70 and TGF-ß were detected by Western blot. B16 tumor-bearing mouse models were generated. The therapeutic effect of the combination of cisplatin (100 µg/mouse i.p., for sequential 3 days) and intratumoral injection of DC cells (3×10(6)/mouse, twice with a 7-day interval) in the tumor-bearing mouse models was evaluated. Expression of MHC II, ICAM-1 and CD86 was analyzed by flow cytometry. The mice were sacrificed at 28 days after tumor cell inoculation. The tumors were removed and weighed, and tissue samples were taken for pathological examination. Tumor infiltrating lymphocytes (TIL) were isolated by discontinuous gradient centrifugation. The distribution of T-reg and CD8(+) T cells in the TIL was analyzed by flow cytometry, and the ratio of CD8(+) T/T-reg was determined. The activity of cytotoxic lymphocytes (CTL) was determined by microcytotoxicity assay. RESULTS: Cisplatin enhanced both the B16 cell apoptosis and HMGB1 expression. After loading with cisplatin-treated cell lysate, the expression of MHC II, ICAM-1 and CD86 on DC cells were (47.5 ± 8.8)%, (35.5 ± 8.3)% and (36.2 ± 9.2)%, respectively. At 28 days after tumor cell inoculation, the tumor weight of the control group was (2.1 ± 0.6) g, that of the cisplatin group was (0.3 ± 0.2) g and that of cisplatin + DC vaccine group was (0.5 ± 0.2) g, showing a significant inhibition of tumor growth (P < 0.01). Furthermore, the CD8(+) T/T-reg ratio and CTL activity in TIL were also significantly enhanced in the tumor-bearing mice treated with cisplatin + DC vaccine. When the effector-to-target ratio was 20:1, 10:1 and 5:1, the CTL activity in the cisplatin + DC vaccine treated mice was (25.0 ± 5.0)%, (22.0 ± 6.0)% and (14.0 ± 4.0)%, respectively, significantly higher than (8.2 ± 3.6)%, (6.7 ± 1.8)% and (3.6 ± 1.9)%, respectively, in the control group (all P < 0.01). CONCLUSION: Cisplatin promotes the anti-tumor effect of DC vaccine by down-regulating T-reg cells and enhancing the CTL activity in tumors.

siRNA against plasminogen activator inhibitor-1 ameliorates bleomycin-induced lung fibrosis in rats.

AIM: Plasminogen activator inhibitor-1 (PAI-1) is involved in the progression of pulmonary fibrosis. The present study was undertaken to examine the effects on pulmonary fibrosis of silencing PAI-1 expression with small interfering RNA (siRNA) and to assess the possible underlying mechanisms. METHODS: Male Wistar rats were subjected to intratracheal injection of bleomycin (BLM, 5 mg/kg, 0.2 mL) to induce pulmonary fibrosis. Histopathological changes of lung tissue were examined with HE or Masson's trichrome staining. The expression levels of α-smooth muscle actin (α-SMA), collagen type-I and type-III, caspase-3, as well as p-ERK1/2 and PI3K/Akt in the lung tissue were evaluated using imunohistochemistry and Western blot analyses. The fibroblasts isolated from BLM-induced fibrotic lung tissue were cultured and transfected with pcDNA-PAI-1 or PAI-1siRNA. The expression level of PAI-1 in the fibroblasts was measured using real time RT-PCR and Western blot analysis. The fibroblast proliferation was evaluated using MTT assay. RESULTS: Intratracheal injection of PAI-1-siRNA (7.5 nmoL/0.2 mL) significantly alleviated alveolitis and collagen deposition, reduced the expression of PAI-1, α-SMA, collagen type-I and collagen type-III, and increased the expression of caspase-3 in BLM-induced fibrotic lung tissue. In consistence with the in vivo results, the proliferation of the cultured fibroblasts from BLM-induced fibrotic lung tissue was inhibited by transfection with PAI-1-siRNA, and accelerated by overexpression of PAI-1 by transfection with pcDNA-PAI-1. The expression of caspase-3 was increased as a result of PAI-1 siRNA transfection, and decreased after transfection with pcDNA-PAI-1. In addition, the levels of p-ERK1/2 and PI3K/Akt in the fibrogenic lung tissue were reduced after treatment with PAI-1siRNA. CONCLUSION: The data demonstrate that PAI-1 siRNA inhibits alveolitis and pulmonary fibrosis in BLM-treated rats via inhibiting the proliferation and promoting the apoptosis of fibroblasts. Suppression ERK and AKT signalling pathways might have at least partly contributed to this process. Targeting PAI-1 is a promising therapeutic strategy for pulmonary fibrosis.

A preliminary study on the activation and antigen presentation of hepatitis B virus core protein virus-like particle-pulsed bone marrow-derived dendritic cells.

Hepatitis B virus core protein virus-like particles (HBc-VLPs) act as a strong immunogen and are suitable for uptake by dendritic cells (DCs), in which they directly promote DC maturation and migration. To illustrate the utility of global proteomic analysis techniques in elucidating the molecular events that are altered in HBc-VLP-pulsed bone marrow-derived DCs (BMDCs) and to gain a better understanding of the molecular mechanisms of capture and processing of HBc-VLP-pulsed BMDCs, an antigen (Ag) delivery system based on HBc-VLP-pulsed BMDCs was developed. Two-dimensional electrophoresis (2-DE) and tandem mass spectrometry (MS/MS) analyses were utilized to analyze the differential protein expression patterns between HBc-VLP-pulsed and untreated BMDCs. Protein spots with significantly altered expression levels were detected, identified and validated. The results showed that exogenous HBc-VLPs were phagocytosed efficiently by BMDCs and enhanced the efficacy of BMDC maturation and Ag presentation, VLPs also induced high levels of Ag-specific CD8(+) T cells that displayed high cytotoxic T lymphocyte (CTL) activity in vivo. Several differentially expressed proteins, including growth factor receptor bound protein 2 (Grb2) and annexin A2 (AnxA2), were detected by proteomic analysis, identified by mass spectrometry and validated by western blot.

Molecular mechanism of cell apoptosis by paclitaxel and pirarubicin in a human osteosarcoma cell line.

BACKGROUND: Paclitaxel and pirarubicin exhibit cytotoxic and antitumor activities. However, little is known about the apoptosis-inducing effects of paclitaxel and pirarubicin on human osteosarcoma MG-63 cells. METHODS: The effects of paclitaxel and pirarubicin on cell cycle arrest and apoptosis were studied in MG-63 cells using flow cytometry. PCNA, Bcl-2, Bax, cyclin D1 and cyclin E expression was assessed by Western blotting. RESULTS: Paclitaxel and pirarubicin caused G2/M and G0/G1 cell cycle arrest in MG-63 cells, respectively. Apoptosis of MG-63 cells mediated by paclitaxel was dependent on treatment duration. Interestingly, in cells treated with pirarubicin, apoptosis was related to treatment duration at concentrations of 10(2)-10(3) nM, whereas the effect of treatment duration was less marked at concentrations >10(4)-10(5) nM. Furthermore, paclitaxel and pirarubicin suppressed the expression of PCNA, cyclin D1, cyclin E and Bcl-2, and increased Bax expression. CONCLUSION: These results suggest that the G2/M or G0/G1 cell cycle arrest and apoptosis induced by paclitaxel and pirarubicin are Bcl-2/Bax dependent, suggesting favorable effects of combination therapy with paclitaxel and pirarubicin in the treatment of osteosarcoma.

[Immunoadjuvant effect of Hsp70L1 in tumor vaccine].

AIM: To explore the immune enhancement of Hsp70L1 in the tumor cell vaccines. METHODS: TRP2(153-243) and Hsp70L1 genes were obtained by RT-PCR from B16 cells in murine melanoma and from spleens of C57BL/6 mice and then were inserted into pcDNA3.1/V5-His eukaryotic expression vectors respectively. The recombinants of pTRP2, pHSP70L1 or pTRP2-Hsp fusion gene were obtained and transfected into B16 cells respectively. TRP2(153-243), HSP70L1 or TRP2-Hsp fusion gene-expressing B16 cells were then induced to necrosis by freezing-thawing or to apoptosis by mitomycin C. C57BL/6 mice were immunized with the necrotic or apoptotic B16 cells twice, then the live B16 tumor cells were transplanted into the immunized mice and the tumor growth was observed in some tumor-bearing mice. IFN-gamma-producing cells in splenocytes were measured by flow cytometry and the CTL activity of spleno-lymphocyte was detected by LDH release assay. RESULTS: After the normal mice were immunized with the necrotic or apoptotic tumor vaccines modified with TRP2(153-243), Hsp70L1 or TRP2-Hsp fusion genes, CTL lysis activity and IFN-gamma production from the splenic lymphocytes were promoted in the groups of Hsp70L1 and TRP2-Hsp modified tumor vaccines (P<0.05 or P<0.01). Additionally, the tumor growth was inhibited obviously in the groups of mice immunized with necrotic tumor vaccines (P<0.05 or P<0.01). However, no marked inhibition of tumor growth was observed in the groups of mice immunized with apoptotic tumor vaccines (P>0.05). CONCLUSION: Hsp70L1 remarkably improves the immunogenicity of B16 tumor vaccines, especially that of necrotic tumor vaccines.

[Immunostimulatory and antitumor effect of Flt3L and CCL5 on DNA vaccine in DNA prime/protein boost strategy].

AIM: To investigate the immune enhancment and antitumor effect of the recombinant plasmids pFlt3L and pCCL5 in DNA prime/protein boost regimens. METHODS: The mice were coimmunized with HBcAg DNA vaccine and the two cytokines DNA constructs by intramuscular injection for three times at an interval of 2 weeks. Then the mice were boosted with HBc particle proteins or DNA vaccines, respectively. The immune efficacy was evaluated by tumor growth curve. To further investigate the mechanism of inhibiting tumor growth, lymphocytes proliferation response and the number of IFN-gamma-producing cells in splenocytes were measured by MTT or flow cytometry, respectively. The levels of IL-2 and IL-4 in supernatant of spleno-lymphocyte cultures were measured by ELISA. The CTL activity of spleno-lymphocyte was detected with LDH release assay. RESULTS: Compared with negative control, DDP/Adj group significantly inhibit tumor growth; splenocytes proliferation response and the numbers of IFN-gamma-producing cells in DDD/Adj group and DDP/Adj group were significantly higher (P<0.05 or P<0.01). The levels of IL-2 in supernatant of spleno-lymphocyte cultures in DDD/Adj group and DDP/Adj group were also markedly higher than that of negative control (P<0.05); but the levels of IL-4 were no differences in all groups (P>0.05). The CTL activities in group of DDS/Adj and DDD/Adj were stronger than that of other groups (P<0.01 or P<0.05). While, the CTL killing activity in DDS/Adj group was over that of DDD/Adj (P<0.01 or P<0.05). CONCLUSION: The significant Th1 response and specific CTL against B16-HBc tumor cells are elicited by the combination of Flt3L and CCL5 in the DNA prime/protein boost strategy.

[Establishment of B16-HLA-MAGE-3 melanoma cell line coexpressing HLA-A 0201/K(b) and MAGE-3 and its role in tumor vaccine evaluation].

AIM: To establish a tumor model in HLA-A2.1 transgenic mice to examine the efficacy of MAGE-3 vaccine, a cell line coexpressing HLA-A 0201/K(b) and MAGE-3 is established. METHODS: B16-HLA-MAGE-3 melanoma was obtained by means of cotransfection of HLA-A 0201/K(b) and MAGE-3 to B16 melanoma. RT-PCR, FCM analysis and Western blot were used to detect the mRNA or protein of HLA-A 0201/K(b) or MAGE-3 expression in B16-HLA-MAGE-3. The ability of MAGE-3 antigen to be processed and presented in the B16-HLA-MAGE-3 cell line were observed by CTL activity detection and tumor challenge test. RESULTS: Transcription and protein expression of HLA-A 0201/H-2k(b) and MAGE-3 were demonstrated in B16-HLA-MAGE-3 cells. CTL activity of splenocytes in immunized mice against B16-HLA-MAGE-3 was detected and the growth of B16-HLA-MAGE-3 in immunized mice was also inhibited. CONCLUSION: MAGE-3 antigen is able to be processed and presented efficiently by B16-HLA-MAGE-3 melanoma cells and this cell can be employed to test HLA-A2 restricted epitope immunogenicity in the A2-transgenic mice.

Augmented humoral and cellular immune responses induced by canine adenovirus type 1 DNA vaccine in BALB/c mice.

Infectious canine hepatitis (ICH) is caused by canine adenovirus type 1 (CAV-1), which severely harms infected animals. Vaccination provides an effective approach to preventing canine infectious diseases. With the objective of exploring a new vaccination strategy that may prevent or cure ICH, we constructed a DNA vaccine, pVAX1-CpG-Loop, and evaluated its immune efficacy. We found that vaccination of BALB/c mice with the DNA vaccine alone, or priming with DNA vaccine and boosting with the Loop protein, resulted in the following: (1) High-level specific antibody (IgG) against CAV-1 was induced; (2) T cell activation was elicited; and (3) neutralizing antibodies were detectable in immunized mice. Collectively, these data indicate that the availability of a DNA vaccine could prevent hepatitis contagiosa canis.

Comparative proteomics analysis to annexin B1 DNA and protein vaccination in mice.

DNA vaccines have been widely reported to elicit both effective humoral and cellular immune responses, but the mechanisms of antigen processing and presentation in DNA immunization is still ambiguous. Aiming to molecular mechanisms involved in DNA immunization, comparative serum proteomics was introduced to discover differentially expressed proteins after different immunizations. Using two-dimensional electrophoresis and matrix-assisted laser desorption ionisation-time-of-flight mass spectrometry, 23 three-fold or greater up-regulated proteins were separated and identified, including 14 from ANXB1 DNA immunized mice and 9 from annexin B1 protein immunized mice. The histocompatibility class I molecule H2-Q10 (HA10_MOUSE) and proteasome activator PA28 alpha-subunit (PSME1_MOUSE) were found up-regulated in ANXB1 DNA immunized mice, which may contribute to the augmented activation of T lymphocytes. These proteins may serve as potential surrogate markers of successful vaccination and provide research targets for molecular mechanisms of vaccinology.

[Anti-tumor effect of monkshood polysaccharide with adriamycin long circulating temperature-sensitive liposome and its mechanism].

AIM: To observe the synergic action of monkshood polysaccharide (MPS) and adriamycin (ADM) long circulating temperature-sensitive liposome (ALTSL) in targeting therapy for H22 tumor-bearing mice and explore the mechanism. METHODS: The anti-tumor activity was evaluated by using the tumor's weight as an index. The life prolongation rate of mice was calculated according to the survival time of the tumor-bearing mice. The killer activity of NK cells and the lymphocyte transformation rate were detected by the LDH release assay and MTT colorimetry, respectively. The apoptosis of tumor cells and the expressions of p53, Fas, Fas-L and caspase-3 were analyzed by flow cytometry. The expressions of IL-2 mRNA and IL-12 mRNA in spleen lymphocytes were determined by RT-PCR. The pathologic changes of tumor, heart, liver and kidney tissues of the tumor-bearing mice were observed under light microscope. RESULTS: Compared with the adriamycin liposome group, the anti-tumor effects were enhanced in (MPS+ALTSL) group with tumor growth inhibitory rate up to 80.4%. The survival time of the tumor-bearing mice in ALTSL and (MPS+ALTSL) groups was significantly prolonged compared with the ADM group (P<0.01). The killer activity of NK cells was higher in ALTSL group than in the NS and ADM groups, and was highest in (MPS+ALTSL) group. The lymphocyte transformation rate of (MPS+ALTSL) group was markedly increased (P<0.01) as compared with the ADM group. The result of RT-PCR indicated that the expressions of IL-2 mRNA and IL-12 mRNA in lymphocytes in the adriamycin long circulating liposome (ALCL) group were significantly higher than those in the ADM group. Expressions of IL-2 mRNA and IL-12 mRNA was much higher in (MPS+ALTSL) group than in ALTSL group. The pathological examination indicated that in (MPS+ALTSL) group, more lymphocytes and monocytes were found in tumor tissue. CONCLUSION: ALTSL can increase the anti-tumor effect and decrease the side-effects (such as the cytotoxicity) of ADM. MPS combined with ALTSL can enhance killer activity of NK cells and transformation of T cells, supporting their synergic anti-tumor effect.

[Synergistic role between rhIL-2 and adriamycin long circulating temperature-sensitive liposome in targeting therapy on tumor].

AIM: To observe the synergistic role between rhIL-2 and adriamycin long circulating temperature-sensitive liposome (ALTSL) in targeting therapy of H22 tumor-bearing mice and explore their anti-tumor mechanism. METHODS: The antitumor activity was evaluated by using the tumor's weight as an index. The prolongation rate of mouse life was calculated according to the survival time of the tumor-bearing mice. The killer activity of NK cells and the lymphocyte transformation rate were detected by the LDH and MTT colorimetry, respectively. The apoptosis of tumor cells and the expression of p53, Fas, Fas-L and Caspase-3 were analyzed by flow cytometry (FCM). The expression of IL-2 mRNA and IL-12 mRNA in splenocytes was determined by RT-PCR. The pathologic changes of tumor, heart, liver and kidney tissues of the tumor-bearing mice were observed under light microscope. RESULTS: The tumoristatic rate of rhIL-2+ALTSL (73.5%) was higher than that of adriamycin liposome (ADML) group (67.0%). The survival time of tumor-bearing mice in ALTSL and rhIL-2+ALTSL groups was significantly extended as compared with the NS group (treated with normal saline) and the free ADM group (P <0.01 or P <0.05). The killer activities of NK cells of ALTSL group and rhIL-2+ALTSL group were higher than those of the NS and free ADM groups, and was highest in rhIL-2+ALTSL group. The lymphocyte transformation rate of ALTSL+rhIL-2 group markedly increased ( P <0.01) as compared with the free ADM group. The result of RT-PCR indicated that the expression of IL-2 mRNA and IL-12 mRNA in splenocytes in the adriamycin long circulating liposome (ALCL) group was significantly higher than that in the free ADM group. The enhancement of rhIL-2+ALTSL on expression of IL-2 mRNA and IL-12 mRNA was much stronger than that of ALTSL alone. The pathological examination indicated that in rhIL-2+ALTSL group, the tumor cells were mostly destroyed, and a large amount of lymphocytes and monocytes were found in tumor tissue. CONCLUSION: ALTSL can increase the anti-tumor effect and decreased the side-effects (such as the cytotoxicity) of ADM. rhIL-2+ALTSL can induce the apoptosis of tumor cells and enhance killer activities of T cells and NK cells. rhIL-2 and ALTSL can synergistically play the antitumor effect.

[Preparation of monoclonal antibody against human c-erbB2 and identification of its specificity].

AIM: To prepare monoclonal antibody(mAb) against human c-erbB2 and identify its specificity. METHODS: The epitope of human c-erbB2 antigen was analyzed by using computer software and a immunodominant epitope at the carboxyl-terminal was selected. A peptide consisting of 13 amino acids was synthesized and coupled with keyholelimpet hemocyanin (KLH), and then it was used to immunize BLAB/c mice. The splenocytes of the immunized mice were fused with Sp2/0 cells routinely and the hybridoma cells were selected by HAT selected culture, indirect ELISA, and immunohistochemical staining, and cloned by limiting dilution. The specificity of the mAb was identified by cross-reaction test and blocking test. RESULTS: A hybridoma cell line SC8C1, stably secreting anti-c-erbB2 mAb was obtained. The mAb SC8C1 could react to breast cancer tissue expressing c-erbB2 molecule but did not react to other c-erbB2-negative cells. The mAb will lose the activity after being blocked with synthesized 13 peptide. CONCLUSION: A anti-c-erbB2 mAb SC8C1 is prepared successfully using synthesized 13 peptide as immunogen.

Genome sequence comparative analysis of long arm and short arm of human X chromosome.

30% of the genes tested on Xp escaped inactivation, whereas less than 3% of the genes on Xq escaped inactivation. To investigate the molecular mechanism involved in the propagation and maintenance of X chromosome inactivation and escape, the long arm and short arm of the X chromosome were compared for RNA binding density. Nucleotide sequences on the X chromosome were divided into 50 kb per segment that was recorded as a set of frequency values of 7-nucleotide (7 nt) strings using all possible 7 nt strings (4(7) = 16 384). 120 genes highly expressed in the tonsil germinal center B cells were selected for calculating the 7 nt string frequency values of all introns (intron 7nt). Intron 7nt was considered RNAs (RNA population) that simulated the total of small RNA fragments in cells. Knowing the 7 nt frequency values of DNA segments and the intron 7nt, we can calculate the binding density of DNA segments to the intron 7nt that was termed as RNA binding density. The RNA binding density was determined by the amount of complement sequences. The more amount of complement sequences, the more density of RNA binding. The RNA binding density simulated the total of small RNA fragments bound to the DNA segment. Several principal characteristics were observed for the first time: (1) The mean value of RNA binding density of DNA segments on Xp was significantly higher than that on Xq ( P < 0.001); (2) The numbers of DNA segments highly binding RNAs were more on Xp than on Xq (P < 0.001); (3) The clusters of RNA highly binding DNA segments were associated with regions in which genes escape inactivation. It has been suggested that RNAs activate genes and the interaction of RNA-DNA in cells are extensive, for example, RNAs increase DNase I sensitivity of DNA, there is plenty of nonprotein-coding RNAs in cells, the binding specificity of DNA-RNA is far higher than that of DNA-protein and the affinity of DNA with RNA is increased, as compared with DNA. The nonrandom properties of distribution of RNA highly binding segments between Xp and Xq, combined with the finding of RNA activating genes, provide a strong evidence that RNA highly binding segments may serve as DNA signals to propagate activation along a chromosome and vice versa, the DNA segments that less bind RNAs may silence the genes.

[The effects of two TCM prescriptions, Tonifying Kidney and Strengthening Vital Energy, on NF-kappaB activing in aged mouse T cells].

AIM: The aim of the present study was to explore the immunologic mechanism of delaying senescence by Strengthening Vital Energy(SVE), Tonifying Kidney (TK) and combined TK and SVE. METHODS: Mice of 20 months were used as senescence model. The effects of the prescriptions on anti-CD3 antibody-induced NF-kappaB activity and the expression of NF-kappaB in T cells from aged mice were analyzed by electrophoretic mobility shift assay (EMSA) and Western blot, respectively. RESULTS: NF-kappaB activity in anti-CD3 antibody-induced T cells from the aged mice was lower than that from young ones. The three prescriptions raised the activity of NF-kappaB in the T cells from the aged mice to certain extent. Combined TK and SVE had no influence on p65 and p50 subunits expressions. CONCLUSION: The increased NF-kappaB activity may be one of mechanisms underlying the improvement of immune response in aged mice by Chinese medicines.

[RNA responsible for conferring a DNase I sensitive structure on albumin gene in assembled chromatin].

Although the set of genes is virtually the same in all tissues,differential gene expression is appeared in cells of different kinds. Differentiation and ageing are associated with regulation of gene expression that is a fundamental mechanism in eukaryotic development and survival. The sensitivity to DNase I of actively transcribed genes seems to be a general phenomenon. The purpose of the study is to test whether RNAs obtained from different organs or cells can enhance susceptibility of albumin gene to DNase I digestion in BALB/c mouse brain chromatin assembled.RNAs extracted from rat liver, lung, kidney, brain, tRNA from yeast and synthesized RNAs (23 nt completed with mouse alb gene) were added to a system of chromatin reconstitution that was achieved by dialysis from high ionic strength solution. Assembled chromatin was digested with DNase I (12.5 microg/mL) at 20 degrees for 1 min, then PCR assay was used to detect the level of albumin gene digested. PCR products (1200 bp) were run on a 6% polyacylamide gel and analyzed by silver stain assay. RNAs from different organs and synthesized RNAs all increased the sensitivity of albumin gene to DNase I attack in mouse assembled chromatin. The effect was more obvious in liver and lung RNAs than in kidney and brain ones. tRNA from yeast did not enhance the sensitivity of albumin gene to DNase I digestion. RNA increased albumin gene sensitivity to DNase I in a dose-dependent manner. We report here for the first time that RNAs can enhance susceptibility of albumin gene to DNase I digestion. The effect is associated with RNA sources or sequences. It is generally agreed that the formation of gene sensitivity to DNase I, by unfolding of a tightly packed chromatin fiber, is the first step in gene activation, then RNAs that recognize complementary DNA sequences may be the specific factors that affect DNA supercoiling and determine the sensitivity of gene to DNase I digestion. Here we describes "RNA Population Gene Activating Model" that gives a logical interpretation of events leading to expression of specific genes during normal development and differentiation, in the same time,explains ageing and oncogenesis. Gene expression in eukaryotic cells requires two level regulations. The first may be controlled by RNAs that locate complementary regions within the genomes and make these regions loosened potentially, and the second is mainly involved in sequence specific and nonspecific proteins by which genomic regions bound by RNAs are unfolded. In eukaryotic cells, RNA fragments cleaved from all transcripts mix together to form "RNA populations" in which the majority is intron RNA. Every type of RNA fragments and its homologous sequences act as a group to form certain concentration in which repetitive sequences are more effective. If it is considered that there are many groups of RNA fragments in a particular cell,then different groups of RNA fragments are presented in dissimilar cell types of differentiation. Between DNA replication and nucleosome formation, RNA fragments in nuclear liquid will compete with DNA for binding to complement regions, then the chromatin regions bound to RNA can not be wrapped to form typical nucleosomes. After DNA doubles and is divided into 2 cells, these regions containing atypical nucleosomes become loose by function of non-histone. Transcriptionally active regions of chromatin are loose conformation but loosened regions are not always transcriptionally active. In every division, cells suffer in the described procedure that genes express RNAs, then RNAs recognize and imprint DNA. There are different RNA populations in different cells so that they imprint different genes, which is the primary mechanism by which same genes have expression distinctness. Since loosened genes are similar to bacterial operator system, factors in environment around cells play roles in inducing different gene expression to form different RNA population, which is the primary reason of cell differentiation. RNA population produced by certain impressions in genome can not imprint to form the same ones, otherwise immortal cells will be emerged, so that this program also controls ageing and oncogenesis.

[Cooperative effect of rhIL-2 and adriamycin-magnetic albumin microsphere targeting therapy on tumor].

AIM: To observe the synergistic inhibitory effects of rhIL-2 and adriamycin magnetic albumin microsphere(ADM-MAM) targeting therapy on tumor and to explore their antitumor mechanism. METHODS: The antitumor activity was observed using the tumor weight as index. The killer activity of natural killer (NK) cells and the lymphocyte transformation were examined by the LDH release assay and MTT colorimetry, respectively. The apoptosis of tumor cells and the expressions of p53,Fas and FasL were examined by flow cytometry. The expressions of IL-2 and IL-12 were determined by RT-PCR. RESULTS: Compared with the control group, in ADM-MAM targeting therapy group and combination therapy (ADM-MAM and rhIL-2 therapy) group, tumor weight, killer activity of NK cells, and the lymphocyte transformation;were obviously decreased; while the expression of Fas, FasL, IL-2 and IL-12 were markedly increased. CONCLUSION: ADM-MAM targeting therapy exerts anti-tumor effects with fewer side effects, which can be enhanced when combined with rhIL-2 therapy. Such synergistic anti-tumor effects may be realized by stimulating the proliferation of T cells and growth of NK cell to enhance cellular immunological function.