• Journal of Life Science
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• v.22 no.7
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• pp.871-879
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• 2012

Polyploids are a potentially important germplasm source in seedless citrus breeding program. Seedlessness is one of the most promising traits of commercial mandarin breeds that mandarin triploid hybrids possess permanently. The formation of new constant triploid hybrids can be recovered through diploid species hybridization from the fusion of divalent gametes at low frequencyor intra-and inter-ploidy crosses. However, extensive breeding work based on small $F_1$ hybrid seeds developed is impossible without a very effective aseptic methodology and ploidy event. In this study, in vitro embryo culture was employed to recover natural hybrids from monoembryonic diploid, open-pollinated mandarin. Flow cytometry was used to determine ploidy level. A total of 10,289 seeds were extracted from 792 fruits having approximately 13 seeds per fruit. Average frequency of small seeds developed was 7.1%, while the average frequency of small seeds per fruit were: 8.9% for 'Clementine' 10.2% for 'Harehime' 2.6% for 'Kamja' 3.1% for 'Pyunkyool' 2.8% for 'Sadookam' and 7.0% for 'Wilking' mandarin. Average size of a perfect seed was $49.52{\pm}0.07mm^2$ ('Clementine') while the small seed measured $7.95{\pm}0.04mm^2$ ('Clementine'), which was about 1/6 smaller than the perfect seed. In total, 731 small seeds were obtained and all of them contained only one embryo per seed. The efficiency of 'Clementine' was 14 times higher than 'Wilking' and more than 109 times higher than 'Pyunkyool'. The basic information on spontaneous polyploidy provides for the hybridization of constant triploids and increases the efficiency of conventional cross.

• Journal of Ginseng Research
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• v.44 no.3
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• pp.442-452
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• 2020

Backgroud: Sleep deprivation (SD) impairs learning and memory by inhibiting hippocampal functioning at molecular and cellular levels. Abnormal autophagy and apoptosis are closely associated with neurodegeneration in the central nervous system. This study is aimed to explore the alleviative effect and the underlying molecular mechanism of stem-leaf saponins of Panax notoginseng (SLSP) on the abnormal neuronal autophagy and apoptosis in hippocampus of mice with impaired learning and memory induced by SD. Methods: Mouse spatial learning and memory were assessed by Morris water maze test. Neuronal morphological changes were observed by Nissl staining. Autophagosome formation was examined by transmission electron microscopy, immunofluorescent staining, acridine orange staining, and transient transfection of the tf-LC3 plasmid. Apoptotic event was analyzed by flow cytometry after PI/annexin V staining. The expression or activation of autophagy and apoptosis-related proteins were detected by Western blotting assay. Results: SLSP was shown to improve the spatial learning and memory of mice after SD for 48 h, accomanied with restrained excessive autophage and apoptosis, whereas enhanced activation of phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin signaling pathway in hippocampal neurons. Meanwhile, it improved the aberrant autophagy and apoptosis induced by rapamycin and re-activated phosphoinositide 3-kinase/Akt/mammalian target of rapamycin signaling transduction in HT-22 cells, a hippocampal neuronal cell line. Conclusion: SLSP could alleviate cognitive impairment induced by SD, which was achieved probably through suppressing the abnormal autophagy and apoptosis of hippocampal neurons. The findings may contribute to the clinical application of SLSP in the prevention or therapy of neurological disorders associated with SD.

• IMMUNE NETWORK
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• v.1 no.3
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• pp.236-243
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• 2001

Background: An immunological approach for aging mechanism appears to be important. Lymphocyte subsets analysis in peripheral blood is widely performed to assess the immune status and to diagnose and monitor various diseases. Some lymphocyte subsets are known to change with age, but only few data about age-related reference ragnes for these subsets in healthy individuals have been reported. So we attempted to report reference ranges for these subsets in each age group and review changes of the results with age for the secondary studies about immune cell function as lymphocyte blast transformation and immunoglobulin gene rearrangement (VDJ) including recombination activating genes (RAG-1 and RAG-2). Methods: Lymphocyte subset analysis was performed on 302 subjects, 189 males and 113 females with age group of all decades of life. Two color direct immunofluorescene flow cytometry (FCM) was done using $Simultest^{TM}$ IMK-Lymphocyte kit (Becton Dickinson, USA), $FACScan^{TM}$ (Becton Dickinson, USA) and $FACSCalibur^{TM}$ (Becton Dickinson, USA). Lymphocyte subsets analysed were T ($CD3^+$) and B cells ($CD19^+$), helper/inducer T ($CD4^+$) and suppressor/cytotoxic T cells ($CD8^+$), helper/suppressor ($CD4^+/CD8^+$) ratio and natural killer (NK) cells ($CD3^-CD16^+/CD56^+$). The absolute numbers of each subset were calculated from total lymphocyte counts. Data collected was analysed using SAS 6.12. A P-value of < 0.05 was considered significant. Results: We reported the counts and percentages of lymphocyte and these subsets in each age group. There were no statistically significant differences between male and female subjects. The percentage of $CD4^+$ T cells, and the count of NK cells did not show the significant difference among the various age groups. The age-related changes observed in our study were as following: 1) a decrease in the percentages of T cells, B cells and $CD8^+$ T cells ; 2) a decrease in the counts of B cells and $CD8^+$ T cells ; 3) an increase in the percentage and count of NK cells ; and 4) an increase in the $CD4^+/CD8^+$ ratio. Conclusion: The characteristics of aging process appeared to be showing a marked decrease of lympocyte subsets T and B cells as well as T8 ($CD8^+$). The age-related increase of the percentage of cells bearing NK marker can be interpreted as a compensatory consequence to cope with the decrease of T cells related to the thymic involution. These changes with age appeared to be for the secondary study about immune cell function as lymphocyte blast transformation and immunoglobulin gene rearrangement.

• Tuberculosis and Respiratory Diseases
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• v.44 no.4
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• pp.756-765
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• 1997

Background : To study the prognosis of patients with lung cancer, many investigators have reported the methods to detect cell proliferation in tissues including PCNA, thymidine autoradiography, flow cytometry and Ki-67. PCNA, also known as cyclin, is a cell related nuclear protein with 36KD intranuclear polypeptide that is maximally elevated in S phase of proliferating cells. In this study, PCNA was identified by paraffin-embedding tissue using immunohistochemistry which has an advantage of simplicity and maintenance of tissue architecture. The variation of PCNA expression is known to be related with proliferating fraction, histologic type, anatomic(TNM) stage, degree of cell differentiation, S-phase fraction and survival rate. We analyzed the correlation between PCNA expression and S-phase fraction, survival. Method : To investigate expression of PCNA in primary lung cancer, we used immunohistochemical stain to paraffin-embedded sections of 57 resected primary non-small cell lung cancer specimen and the results were analyzed according to the cell type, cell differentiation, TNM stage, S-phase fraction and survival. Results : PCNA expression was divided into five group according to degree of staging(-, +, ++, +++, ++++). Squamous cell type showed high positivity than in adenocarcinoma. Nonsignificant difference related to TNM stage was noticed. Nonsignificant difference related to degree of cell differentiation was noticed. S-phase fraction was increased with advance of PCNA positivity, but it could not reach the statistic significance. The 2 year survival rate and median survival time were -50% 13 months, +75% 41.3 months, ++73% 33.6 months, +++67% 29.0 months, ++++25% 9 months with statistic significance (P<0.05, Kaplan-Meier, generalized Wilcox). Conclusion : From this study, PCNA expression was high positive in squamous cell cancer. And, there was no relationship between PCNA positivity and TNM stage, cellular differentiation or S-phase fraction. But, the patients with high positive PCNA staining showed poor survival rate than the patients with lower positive PCNA staining (p<0.05). It was concluded that PCNA immunostaining is a simple and useful method for survival prediction in paraffin embedded tissue of non-small cell lung cancer.

• Clinical and Experimental Pediatrics
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• v.52 no.9
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• pp.1015-1020
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• 2009

Purpose:The aim of this study is to explore the effect of the Toll-like receptor 9 (TLR9) expressed in plasmacytoid dendritic cells (pDCs) that respond to antigen to Th2 immune deviation in allergic patients. Methods:Subjects consisted of 19 allergic patients and 17 healthy volunteers. Skin prick tests and nasal provocation tests were performed for the two groups. Peripheral blood mononuclear cells (PBMCs) were collected from subjects and analyzed for the Lineage Cocktail (CD3, CD14, CD16, CD19, CD20, CD56) (-), HLA-DR (+), and CD123 (+) using flow cytometry. In addition, we analyzed TLR9 mRNA by reverse transcriptase-polymerase chain reaction. The level of $interferon-{\alpha}$ ($IFN-{\alpha}$) of the PBMCs following stimulation with the TLR9 ligand CpG-ODN 2216 was also evaluated. Results:Analyses of CD123 (+) revealed a nearly similar distribution for the classical pDC markers in the allergic group ($0.1%{\pm}0.04%$) and in the controls ($0.25%{\pm}0.23%$). The mRNA levels of TLR9 on PBMCs were not different between the allergic group and the controls ($1.29{\pm}0.41$ vs. $1.25{\pm}0.23$, respectively). Additionally, the level of $IFN-{\alpha}$ in PBMCs exposed to stimuli of the TLR9 ligand CpG-ODN 2216 was not significantly different between the two groups ($911{\pm}829$ vs. $1,095{\pm}888pg/mL$, respectively). Conclusion:We found no evidence that TLR9-dependent immune responses in human pDCs are associated with allergic status.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.23
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• pp.10407-10412
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• 2015

Background: ${\beta}$-elemene, extracted from herb medicine Curcuma wenyujin has potent anti-tumor effects in various cancer cell lines. However, the activity of ${\beta}$-elemene against glioma cells remains unclear. In the present study, we assessed effects of ${\beta}$-elemene on human glioma cells and explored the underlying mechanism. Materials and Methods: Human glioma U87 cells were used. Cell proliferation was determined with MTT assay and colony formation assay to detect the effect of ${\beta}$-elemene at different doses and times. Fluorescence microscopy was used to observe cell apoptosis with Hoechst 33258 staining and change of glioma apoptosis and cell cycling were analyzed by flow cytometry. Real-time quantitative PCR and Western-blotting assay were performed to investigated the influence of ${\beta}$-elemene on expression levels of Fas/FasL, caspase-3, Bcl-2 and Bax. The experiment was divided into two groups: the blank control group and ${\beta}$-elemne treatment group. Results: With increase in the concentration of ${\beta}$-elemene, cytotoxic effects were enhanced in the glioma cell line and the concentration of inhibited cell viability ($IC_{50}$) was $48.5{\mu}g/mL$ for 24h. ${\beta}$-elemene could induce cell cycle arrest in the G0/G1 phase. With Hoechst 33258 staining, apoptotic nuclear morphological changes were observed. Activation of caspase-3,-8 and -9 was increased and the pro-apoptotic factors Fas/FasL and Bax were upregulated, while the anti-apoptotic Bcl-2 was downregulated after treatment with ${\beta}$-elemene at both mRNA and protein levels. Furthermore, proliferation and colony formation by U87 cells were inhibited by ${\beta}$-elemene in a time and does-dependent manner. Conclusions: Our results indicate that ${\beta}$-elemene inhibits growth and induces apoptosis of human glioma cells in vitro. The induction of apoptosis appears to be related with the upregulation of Fas/FasL and Bax, activation of caspase-3,-8 and -9 and downregulation of Bcl-2, which then trigger major apoptotic cascades.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.1
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• pp.467-474
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• 2014

Objective: To observe the effects of allogeneic and autologous transfusion on cellular immunity, humoral immunity and secretion of serum inflammatory factors and perforin during the perioperative period in patients with malignant tumors. Methods: A total of 80 patients (age: 38-69 years; body weight: 40-78 kg; ASA I - II) receiving radical operation for gastro-intestinal cancer under general anesthesia were selected. All the patients were divided into four groups based on the methods of infusion and blood transfusion: blank control group (Group C), allogeneic transfusion group (group A), hemodiluted autotransfusion Group (Group H) and hemodiluted autotransfusion + allogenic transfusion Group (A+H group). Venous blood was collected when entering into the surgery room ($T_0$), immediately after surgery ($T_1$) and 24h ($T_2$), 3d ($T_3$) and 7d ($T_4$) after surgery, respectively. Moreover, flow cytometry was applied to assess changes of peripheral blood T cell subpopulations and NK cells. Enzyme linked immunosorbent assays were performed to determine levels of IL-2, IL-10, TNF-${\alpha}$ and perforin. Immune turbidimetry was employed to determine the changes in serum immunoglobulin. Results: Both CD3+ and NK cells showed a decrease at $T_1$ and $T_2$ in each group, among which, in group A, CD3+ decreased significantly at $T_2$ (P<0.05) compared with other groups, and CD3+ and NK cell reduced obviously only in group A at $T_3$ and $T_4$ (P<0.05). CD4+ cells and the ratio of D4+/CD8+ were decreased in groups A, C and A+H at $T_1$ and $T_2$ (P<0.05). No significant intra- and inter-group differences were observed in CD8+ of the four groups (P<0.05). IL-2 declined in group C at $T_1$ and $T_2$ (P<0.05) and showed a decrease in group A at each time point (P<0.05). Moreover, IL-2 decreased in group A + H only at $T_1$. No significant difference was found in each group at $T_1$ (P<0.05). More significant decrease in group ?? at $T_2$, $T_3$ and $T_4$ compared with group A (P<0.05), and there were no significant differences among other groups (P>0.05). IL-10 increased at $T_1$ and $T_2$ in each group (P<0.05), in which it had an obvious increase in group A, and increase of IL-10 occurred only in group A at $T_3$ and $T_4$ (P<0.05). TNF-${\alpha}$ level rose at $T_1$ (P<0.05), no inter- and intra-group difference was found in perforin in all groups (P<0.05). Compared with the preoperation, both IgG and IgA level decreased at $T_1$ in each group (P<0.05), and they declined only in Group A at $T_2$ and $T_3$ (P<0.05), and these parameters were back to the preoperative levels in other groups. No significant differences were observed between preoperative and postoperative IgG and IgA levels in each group at $T_4$ (P>0.05). No obvious inter- and intra-group changes were found in IgM in the four groups (P>0.05). Conclusions: Allogeneic transfusion during the perioperative period could obviously decrease the number of T cell subpopulations and NK cells and the secretion of stimulating cytokines and increase the secretion of inhibiting cytokines in patients with malignant tumors, thus causing a Th1/Th2 imbalance and transient decreasing in the content of plasma immune globulin. Autologous transfusion has little impact and may even bring about some improvement oo postoperative immune function in patients with tumors. Therefore, cancer patients should receive active autologous transfusion during the perioperative period in place of allogeneic transfusion.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.12
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• pp.4773-4780
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• 2014

Background: To investigate the effects of small interference RNA (siRNA) targeting BCR/ABL mRNA on proliferation and apoptosis in the K562 human chronic myeloid leukemia (CML) cell line and to provide a theoretical rationale and experimental evidence for its potential clinical application for anti-CML treatment. Materials and Methods: The gene sequence for BCR/ABL mRNA was found from the GeneBank. The target gene site on the BCR/ABL mRNA were selected according to Max-Planck-Institute (MPI) and rational siRNA design rules, the secondary structure of the candidate targeted mRNA was predicted, the relevant thermodynamic parameters were analyzed, and the targeted gene sequences were compared with BLAST to eliminate any sequences with significant homology. Inhibition of proliferation was evaluated by MTT assay and colony-formation inhibiting test. Apoptosis was determined by flow cytometry (FCM) and the morphology of apoptotic cells was identified by Giemsa-Wright staining. Western blotting was used to analyze the expression of BCR/ABL fusion protein in K562 cells after siRNA treatment. Results: The mRNA local secondary structure calculated by RNA structure software, and the optimal design of specific siRNA were contributed by bioinformatics rules. Five sequences of BCR/ABL siRNAs were designed and synthesized in vitro. Three sequences, siRNA1384, siRNA1276 and siRNA1786, which showed the most effective inhibition of K562 cell growth, were identified among the five candidate siRNAs, with a cell proliferative inhibitory rate nearly 50% after exposure to 12.5nmol/L~50nmol/L siRNA1384 for 24,48 and 72 hours. The 50% inhibitory concentrations ($IC_{50}$) of siRNA1384, siRNA1276 and siRNA1786 for 24hours were 46.6 nmol/L, 59.3 nmol/L and 62.6 nmol/L, respectively, and 65.668 nmol/L, 76.6 nmol/L, 74.4 nmol/L for 72 hours. The colony-formation inhibiting test also indicated that, compared with control, cell growth of siRNA treated group was inhibited. FCM results showed that the rate of cell apoptosis increased 24 hours after transfecting siRNA. The results of annexinV/PI staining indicated that the rate of apoptosis imcreased (1.53%, 15.3%, 64.5%, 57.5% and 21.5%) following treamtne with siRNAs (siRNA34, siRNA372, siRNA1384, siRNA1276 and siRNA1786). Morphological analysis showed td typical morphologic changes of apoptosis such as shrunken, fragmentation nucleus as well as "apoptotic bodies" after K562 cell exposure to siRNA. Western blot analysis showed that BCR/ABL protein was reduced sharply after a single dose of 50nmol/L siRNA transfection. Conclusions: Proliferation of K562 cells was remarkbly inhibited by siRNAs (siRNA1384, siRNA1276 and siRNA1786) in a concentration-dependent manner in vitro, with effective induction of apoptosis at a concentration of 50 nmol/L. One anti-leukemia mechanism in K562 cells appeared that BCR/ABL targeted protein was highly down-regulated. The siRNAs (siRNA1384, siRNA1276 and siRNA1786) may prove valuable in the treatment of CML.

• Clinical and Experimental Pediatrics
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• v.45 no.2
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• pp.247-255
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• 2002

Purpose : This study was undertaken to obtain basic data about the megakaryocyte colony formation of fetal liver cells by using immunocytochemical staining and ex vivo culture with growth factors. Methods : The mononuclear cells were isolated from fetal liver and bone marrow with idiopathic thrombocytopenic purpura(ITP) and pancytopenia. These mononuclear cells were cultured in $MegaCult^{TM}-C$(Stem Cell Tech, Canada) media in the presence of growth factors and CFU-Megakaryocyte( CFU-Mk) colonies were counted on day 12. The expansion of CD34+ and CD41+ cell was analyzed by flow cytometry after 5 days incubation using flask culture. Results : The numbers of CFU-Mk colonies of mononuclear cells obtained from fetal liver in the 11th week gestational age were more than those in the 19th week specimens; growth factors could not enhance the colony expansion in all cases. Total numbers of CFU-Mk colony of fetal liver cells were higher than bone marrow from ITP or pancytopenia groups. The numbers of pure or large CFU-Mk colonies of fetal liver cells were also higher than bone marrow specimens. The rate of CD34+ cell expression of fetal liver was increased after flask culture and the enhancement effect of epression was seen only in cases which added thrombopoietin. The rate of CD41+ cell expression of fetal liver was increased after incubation, but the enhancement effect of growth factors was unclear. Conclusion : This study revealed good results about the megakaryocyte colony assay of fetal liver mononuclear cells using $MegaCult^{TM}-C$ media. This study suggests that the fetal liver could be a good source of megakaryocytic progenitor cells for clinical application in hematopoietic stem cell transplantation.

• Microbiology and Biotechnology Letters
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• v.41 no.4
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• pp.425-432
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• 2013

Typha orientalis, also known as bulrush or cattail, is a perennial herbaceous plant found in freshwater wetlands and has been widely used in constructed wetlands for wastewater treatment. Recent data has revealed that SH21B, a mixture composed of seven herbs including T. orientalis, exhibited an anti-adipogenic activity by the inhibition of the expression of adipogenic regulators. However, the anti-cancer effect of T. orientalis and its molecular mechanisms remain unclear. In this study, we evaluated the anti-cancer effect and its mechanism in the methanol extract of T. orientalis (METO) on human colon carcinoma HT29 cells. It was found that METO treatment showed cytotoxic activity in a dose-dependent manner, and induced G2/M cell cycle arrest and apoptosis in HT29 cells. The induction of G2/M arrest by METO was associated with the up-regulation of phospho-Cdc2 (Tyr15), an inactive form of Cdc2 and the down-regulation of Cdc25c phosphatase. METO also induced tumor suppressor p53 and cyclin-dependent kinase inhibitor p21 (WAF1/CIP1) expression. In addition, METO-induced apoptosis was characterized by the proteolytic activation of caspase-3, degradation of poly ADP ribose polymerase (PARP), and up-regulation of death receptor FAS and pro-apoptotic Bax expression. Collectively, these results indicate that the cell cycle inhibition and apoptosis induction of METO in HT29 cells allows for the possibility of its use in anti-cancer therapies.