Cloning and Expression Patterns of Coumarate-3-hydroxylase Gene from Angelica sinensis and Its Correlation with Content of Ferulic Acid / 中国实验方剂学杂志

ObjectiveTo clone coumarate-3-hydroxylase gene （C3H） from Angelica sinensis， and analyze the correlation between its bioinformatics， expression patterns and content of ferulic acid， and to explore the functions of ASC3H. MethodReal-time polymerase chain reaction （Real-time PCR） was used to clone the full-length cDNA of ASC3H based on the transcriptome dataset of A. sinensis， and the bioinformatics analysis of the gene sequence was carried out. Real-time PCR and high performance liquid chromatography （HPLC） were used to determine relative expression of ASC3H and content of ferulic acid in different root tissues of A. sinensis （periderm， cortex and stele）. ResultThe open reading frame （ORF） of ASC3H （GenBank accession number： MN2550298） was 1 530 bp， encoding 509 amino acids， with a theoretical molecular weight of 57.86 kDa and an isoelectric point of 8.36. It was a hydrophilic protein that was located in the chloroplast with multiple phosphorylation sites and a transmembrane region， and contained a conserved domain CGYDWPKGYGPIINVW_P450 （383-399 aa） in cytochrome P450. Multiple amino acid sequence alignment analysis showed that ASC3H had high similarity with C3H from other plants， especially Ammi majus in Umbelliferae. The Real-time PCR revealed that ASC3H had different expressions in periderm， cortex and stele tissues of A. sinensis roots. It was found from HPLC that the cortex tissues had the highest content of ferulic acid， and the stele tissues had the lowest. ConclusionASC3H was successfully cloned from A. sinensis， and its sequence characteristics were understood more clearly， suggesting that ASC3H might be involved in the ferulic acid biosynthesis pathway of A. sinensis. This paper provided a basis for further studying the functions of the gene and exploring the biosynthesis and regulation mechanism of ferulic acid in A. sinensis， while laying the foundation for the genetic improvement of A. sinensis．

Porcine Circular RNA Insulin-like Growth Factor 1 Receptor Promotes Adipocyte Differentiation / 中国生物化学与分子生物学报

Circular RNA (circRNA), as a competitive endogenous RNA (ceRNA), plays an importantrole in the regulation of cell differentiation. The purpose of this study was to identify and analyze porcinecircular RNA insulin-like growth factor 1 receptor (circIGF1R), explore its expression patterns, construct a ceRNA regulatory network related to circIGF1R, and explore the regulation of its ectopicexpression on adipogenic differentiation of mouse mesenchymal stem cells (C3H10T1 / 2) effect. Forwardand reverse PCR, Sanger sequencing, RNase R enzyme digestion tests, and qRT-PCR were used toverify that circIGF1R is a circRNA formed by the second exon of insulin-like growth factor 1 receptor(IGF1R). It was expressed in all tissues of pigs, and its expression level increased with age in adiposetissues. miRDB, TargetScan and miRWalk online software were used to predict circIGF1R target genes. RNAhybrid software was used for binding site prediction. DAVID bioinformatics functional analysissoftware was used to perform GO and KEGG enrichment analysis on candidate target genes. Cytoscapesoftware was used to construct the ceRNA network diagram. Based on the gene expression correlation andpredicted target relationship, the GO and KEGG enrichment analysis was drawn and the ceRNA networkwas constructed; the dual luciferase reporter gene test was used, and we found that circIGF1R andFABP4 can bind to ssc (Sus scrofa chromosome) -miR-133a-5p. The circIGF1R overexpression vectorwas successfully constructed and expressed in C3H10T1/ 2 cells. It was found that after overexpression ofcircIGF1R, the expression of key adipogenic regulatory factors CEBPa, CEBPß, FABP4 and PPAR? increased significantly(P<0. 01), and the number of lipid droplets increased significantly. The results ofthis study show that circIGF1R exists in pig adipose tissues, and may positively regulate the adipogenicdifferentiation of C3H10T1/ 2 cells through the ceRNA mechanism, which lays a theoretical foundation forfurther research on circIGF1R regulating the adipogenic differentiation of pig precursor intramuscularadipocytes.

Role of zinc finger protein 36, C3H type-like 1 mediating astrocytes activation in motor neuron degeneration in amyotrophic lateral sclerosis / 解剖学报

Objective To investigate the role of zinc finger protein 36,C3H type-like 1 (ZFP36L1) mediating astrocytes activation in the degeneration of motor neurons in amyotrophic lateral sclerosis (ALS). Methods Superoxide dismutase 1 (S0D1)-G93A transgenic mice were used as animal models, the wild-type littermates as the control (13 mice were taken from mutant and wild-type mice at each time point) . The ZFP36L1 mRNA and protein levels of the spinal cord in the early, middle and late stage were detected by Real-time PGR and Western blotting. The expression and distribution of ZFP36L1 in the spinal cord were detected by immunofluorescence. Primary astrocyte model was established from 15 postnatal 1-2 day mice. The ZFP36L1 mRNA and protein levels in astrocytes were detected by Real-time PCR and Western blotting. Si-ZFP36L1 was transfected into SOD1-G93A mutant primary astrocytes. The transfection efficiency was detected by Western blotting. Tumor necrosis factor a (TNF-a) and interleukin-18 (IL-18) secreted from astrocytes after transfection were assessed by Western blotting and ELISA. After silencing ZFP36L1 in SOD1-G93A mutant primary astrocytes, it was cocultured with SOD1-G93A mutant NSC34 cells. 5 ' -ethynyl-2' deoxyuridine (EdU) test and the level of proliferating cell nuclear antigen (PCNA) were used to determine the effect of ZFP36L1 on NSC34 cell proliferation. TUNEL test and the level of cleaved-Caspase-3 were used to determine the effect of ZFP36L1 on NSC34 cell apoptosis. Blank small interfering RNA(siRNA) was transfected as the control group. Results Compared with the wild-type mice, the mRNA and protein levels of ZFP36L1 were downregulated in the spinal cord of SOD1-G93A transgenic mice. In wild type mice, ZFP36L1 positive cells were mainly [

MYOZ2 Promoted Adipogenic Differentiation of C3H10T1 / 2 Cells by Negatively Regulating the Expression of TCAP / 中国生物化学与分子生物学报

The objective of this study was to investigate the expression profile of the myozenin2 (MYOZ2) gene and elucidate its effect on adipogenic differentiation of C3H10T1 / 2 cells and its possible mechanism∙ The longissimus dorsi‚ subcutaneous fat and liver tissue was collected from 180-day-old Mashen pigs‚ 60-day-old ICR mice‚ 35-day-old Ross broiler and 12-month-old Small tail han sheep‚ and the expression profile of the MYOZ2 gene mRNA was detected∙ The results showed that the MYOZ2 gene has similar patterns of tissue expression in examined species‚ with the highest expression level in longissimus dorsi‚ and a small amount of expression in the subcutaneous fat and liver tissue∙ After the MYOZ2 gene was silenced in C3H10T1 / 2 cells‚ qRT-PCR results showed that the expression levels of key adipogenic genes PPARγ and FABP4 were significantly down-regulated compared with the control group (P < 0∙ 01) ; Western Blotting results showed that the PPARγ protein content was significantly decreased (P < 0∙ 05) ; Oil red O staining showed that the number of lipid droplets and the content of triglyceride were significantly decreased after silencing MYOZ2 (P < 0∙ 05) ∙ The expression of fatty acid metabolism related genes SCD‚ FASN‚ SREBP1‚ NR1H3‚ DGAT1‚ PNPLA2‚ HSL‚ CES1‚ CPT1 after MYOZ2 silencing were detected by qRT-PCR∙ The results showed that SCD‚ FASN‚ SREBP1‚ PNPLA2 and HSL were significantly down-regulated (P < 0∙ 01) ‚ NR1H3 was significantly reduced (P < 0∙ 05) ‚ DGAT1 expression was down-regulated but the difference was not significant‚ CES1 and CPT1 were significantly up-regulated (P < 0∙ 05) ∙ The STRING database was used to construct a MYOZ2-related protein interaction network map‚ and it was found that MYOZ2 may affect the adipogenic differentiation through the interaction of titin-cap (TCAP) and PPARγ∙ After silencing TCAP‚ qRT-PCR results showed that compared with the control group‚ the expression of key adipogenic genes PPARγ and FABP4 were significantly up-regulated (P < 0∙ 01) ; Western Blotting results showed that PPARγ protein was significantly increased (P< 0∙ 05) ; Oil red O staining showed that the number of lipid droplets and the content of triglyceride were significantly increased after TCAP silencing (P < 0∙ 05) ∙ qRT-PCR was used to detect the expression of TCAP after silencing MYOZ2‚ and the results showed that the expression of TCAP was significantly increased (P<0∙ 01) ∙ In summary‚ MYOZ2 was highly expressed in longissimus dorsi and lower expressed in subcutaneous fat and liver tissues∙ In addition‚ MYOZ2 may regulate the expression of key adipogenic genes PPARγ and FABP4 through the interaction of MYOZ2-TCAP -PPARγ‚ and to further regulate the expression of fatty acid metabolism related genes SCD‚ FASN‚ SREBP1‚ NR1H3‚ DGAT1‚ PNPLA2‚ HSL‚ CES1 and CPT1‚ thus playing an important role in the process of adipogenic differentiation∙

FNDC5 Regulates the Adipogenic Differentiation of C3H10T1/2 Cells by Inhibiting the Phosphorylation of ERK1/2 / 中国生物化学与分子生物学报

The aim of this study was to explore the regulatory mechanism of Type Ⅲ domain-containing protein5 (FNDC5) on adipogenic differentiation in C3H10T1/2 cells. qRT-PCR and Western blot were used to detect the expression of FNDC5 during adipogenic differentiation of C3H10T1/2 cells. The lentivirus-coated overexpression and interference vector of FNDC5 were constructed and transfected into C3H10T1/2 cells. qRT-PCR was used to detect the expression of the key genes of adipogenic differentiation. Oil red O staining was used to detect the formation of lipid droplets; Western blot was used to detect the content of ERK1/2 and ERK1/2 phosphorylated protein (P-ERK1/2). After 8 days of adipogenic differentiation of C3H10T1/2 cells, the expression of Fndc5 increased significantly. After overexpression of FNDC5 in C3H10T1/2 cells, the expression of key genes for adipogenic differentiation, including peroxisome proliferator-activated receptor-酌 (PPAR酌), CCAAT enhancer binding protein beta (C/EBP茁), fatty acid binding protein 4 (FABP4) and CCAAT enhancer binding protein alpha (C/EBPα), all decreased significantly. The content of lipid droplets and P-ERK1/2 also decreased significantly. On the contrary, after interference of FNDC5 in C3H10T1/2 cells, the expression of key genes for adipogenic differentiation, including PPARγ, C/EBP茁, FABP4 and C/EBPα were significantly increased. Meanwhile, the content of lipid droplets and P-ERK1/2 also increased significantly. This study found that FNDC5 can inhibit the adipogenic differentiation of C3H10T1/2 cells by inhibiting the phosphorylation level of ERK1/2, which can provide reference data for the mechanism of FNDC5 in regulating fat deposition.

Cloning and Expression Patterns of LmC3H1 Gene in Lonicera macranthoides and Its Correlation with Chlorogenic Acid Content / 中国实验方剂学杂志

Objective::To clone p-coumaroyl quinate/shikimate 3' -hydroxylase gene from Lonicera macranthoides, and analyze its bioinformatics and expression patterns with chlorogenic acid content, in order to speculate the functions of LmC3H1 gene from L. macranthoides. Method::The full-length cDNA sequence of LmC3H1 gene was cloned by reverse trascription polymerase chain reaction(RT-PCR) and RACE techniques. The bioinformatics analysis of the gene sequence was carried out by using relevant software.Real-time fluorescence quantification PCR(Real-time PCR) and HPLC were used to determine relative expression of LmC3H1 and content of chlorogenic acid in stems, leaves and flowers of different flowering stages. Result::The LmC3H1 (GenBank: MN177695) gene was cloned, and the open reading frame (ORF) of it was 1 533 bp in length and encoded 510 amino acids. The molecular formula was C2618H4134N718O727S22, the relative molecular mass was 58 005.32, and the isoelectric point was 8.92.It was a hydrophilic protein located in the chloroplast with a transmembrane region LLLIPAVLFLISLVYPLI, and contained a conserved domain CYTOCHROME_P450(433-422 aa) in cytochrome P450.The results of Real-time PCR showed that LmC3H1 was expressed in different degrees in stems, leaves and different flowering stages of L. macranthoides. In the flower development stage, the relative expression of white bud stage was the highest, followed by flower buds and white flowering stage. The ratio of flower to stem and leaf was the highest, and the relative expression of flower was the highest. The HPLC results showed that the content of chlorogenic acid increased from greenish white to golden yellow in flowering stage and golden yellow flowering stage. Among the different organs, the flower had the highest chlorogenic acid, and the stem showed the lowest. Conclusion::The LmC3H1 gene of L. macranthoides is cloned, suggesting that LmC3H1 might be involved in the biosynthesis of L. macranthoides chlorogenic acid. This study provides a basis for further studying the functions of the gene and exploring the biosynthesis and regulation mechanism of L. macranthoides chlorogenic acid, while laying the foundation for the genetic improvement of L. macranthoides.

Cloning and expression of key enzyme genes for chlorogenic acid synthesis in Chrysanthemum morifolium / 中草药

To investigate the effects of the expression of coumaroylquinate 3’-monooxygenase (C3’H) and shikimate O-hydroxycinnamoyltransferase (HCT) in the chlorogenic acid-producing pathway and active ingredients in Chrysanthemum morifolium under flooding stress, we cloned two C3’H genes which were CmC3’H1 and CmC3’H2 and two HCT genes which were CmHCT1 and CmHCT2 by the RT-PCR from Hangju and conducted bioinformatics analysis. During the flower bud differentiation stage, we flooded the C. morifolium and then used β-actin as the reference gene to detect the relative expression of the four genes by the qRT-PCR. Finally, the content of downstream products and other indicators of these four genes in C. morifolium were measured by HPLC. We obtained the four genes’ complete open reading frame and predicted the relative molecular mass of the amino acid sequence and the theoretical isoelectric point (pI). And the protein tertiary structure models were constructed. The qRT-PCR results showed that flooding the C. morifolium for 3 days during the flower bud differentiation stage resulted in significant expression changes of the four genes at different growth stages. The results of HPLC showed that chlorogenic acid, the downstream product catalyzed by the C3’H and the HCT, was significantly higher than that in the control group. It was also found that the content of luteoloside and 3,5-O-di-caffeoylquinic acid was also significantly higher than those of the control group. Therefore, C. morifolium regulates the synthesis of downstream products by regulating the expression of the four genes under flooding stress, thereby responding to flooding stress. And the flooding stress during flower bud differentiation can significantly enhance the accumulation of active ingredients of C. morifolium.

Overexpression of TLR2,TLR4 and MyD88 is associated with inflammation in C3H mice during Chlamydia respiratory infection / 中国免疫学杂志

Objective:To investigate the mechanism of excessive inflammation in the lung of C3H/HeN(C3H) mice following Chlamydia muridarum(Cm) airway infection.Methods:Chlamydial pneumonitis was induced in C3H and C57BL/6(C57) mice by intranasal inoculation with 1×103IFU (inclusion forming unites) of Cm strains.The expression of TLR2,TLR4 and MyD88 mRNA in the lung at different time point post-infection was measured by RT-PCR.Results:Cm infection induced Toll-like receptors expression in two strains of mice.The expression of TLR2 and TLR4 mRNA,especially TLR2 mRNA(P<0.001 or P<0.05),were significantly higher in highly susceptible C3H mice on day 7 and day 14 d post-infection compared with C57 mice.Further studies showed that the expression of MyD88 mRNA was also significantly higher in C3H mice on day 7 post-infection,and maintained high expression untill the day 14.Conclusion:Cm lung infection induced high level of TLR2,TLR4 and MyD88 mRNA expression in C3H mice,which may associate with excessive inflammation in C3H mice.

Antibody Production, Anaphylactic Signs, and T-Cell Responses Induced by Oral Sensitization With Ovalbumin in BALB/c and C3H/HeOuJ Mice

PURPOSE: Two mouse strains, BALB/c and C3H/HeOuJ, broadly used in the field of food allergy, were compared for the evaluation of the allergenic potential of ovalbumin (OVA). METHODS: Sensitization was made by administering 2 different OVA doses (1 and 5 mg), with cholera toxin as Th2-polarizing adjuvant. Antibody levels, severity of anaphylaxis, and Th1 and Th2 responses induced by the allergen were assessed. In addition, because the mice selected had functional toll-like receptor 4, the influence of contamination with lipopolysaccharide (LPS) on the immunostimulating capacity of OVA on spleen cells was also evaluated. RESULTS: Both strains exhibited similar susceptibility to OVA sensitization. The 2 protein doses generated similar OVA-specific IgE and IgG1 levels in both strains, whereas C3H/HeOuJ mice produced significantly more IgG2a. Oral challenge provoked more severe manifestations in C3H/HeOuJ mice as indicated by the drop in body temperature and the severity of the anaphylactic scores. Stimulation of splenocytes with OVA led to significantly higher levels of Th2 and Th1 cytokines in BALB/c, and these were less affected by protein contamination with LPS. CONCLUSIONS: The antibody and cytokine levels induced by OVA in BALB/c mice and the observation that BALB/c spleen cell cultures were more resistant than those of C3H/HeOuJ mice to the stimulus of LPS make this strain prone to exhibit Th2-mediated food allergic reactions and very adequate for the study of the features of OVA that make it allergenic.

C3H/He Mice as an Incompatible Cholangiocarcinoma Model by Clonorchis sinensis, Dicyclanil and N-Nitrosodimethylamine

Clonorchis sinensis is a Group-I bio-carcinogen, associated with cholangiocarcinoma (CCA). The hamster is the only experimental model of C. sinensis-mediated CCA, but we oblige another animal model. The present study intended to develop a C. sinensis (Cs) mediated CCA model using C3H/He mice, co-stimulated with N-nitrosodimethyl-amine (NDMA) and dicyclanil (DC). The mice were divided into 8 groups with different combinations of Cs, NDMA, and DC. Six months later the mice were sacrificed and subjected to gross and histopathological examination. The body weights were significantly reduced among the groups treated with 2 or more agents (eg. Cs+NDMA, Cs+DC, NDMA+DC, and Cs+NDMA+DC). In contrast, liver weight percentages to body weight were increased in above groups by 4.1% to 4.7%. A Change of the spleen weight was observed only in Cs+NDMA group. Though C. sinensis infection is evident from hyperplastic changes, only 1 worm was recovered. T wo mice, 1 from Cs and the other from Cs+DC group, showed mass forming lesions; 1 (281.2 mm3) from the Cs group was a hepatocellular adenoma and the other (280.6 mm3) from the Cs+DC group was a cystic mass (peliosis). Higher prevalence of gray-white nodules was observed in Cs group (42.9%) followed by Cs+NDMA+DC group (21.4%). The mice of the Cs+NDMA+DC group showed hyper-proliferation of the bile duct with fibrotic changes. No characteristic change for CCA was recognized in any of the groups. In conclusion, C3H/He mice produce no CCA but extensive fibrosis when they are challenged by Cs, NDMA, and DC together.

Repair of staphylococcus aureus-infected wound with gene-modified C3H10T1/2 cells expressing BPI-BD3 fusion antibiotic peptide / 解放军医学杂志

Objective To study the antibacterial and tissue reparative effect of BPI-BD3 gene-modified mesenchymal stem cells in a mouse model of wound infection. MethodsC3H10T1/2 cells were transfected with recombinant adenovirus vector pAdxsi-BPI-BD3, the expression of BPI-BD3 fusion protein was verified by RT-PCR and Western blotting. Excision wound with a diameter of 1cm was inoculated with Staphylococcus aureus was made on the back of 30 mice. The mice were randomly divided into 3 groups (10 each). Mice in group T were injected with BPI-BD3 gene-modified C3H10T1/2 cells through caudal vein, those in group C were injected with unmodified C3H10T1/2 cells, and in group N were injected with PBS as control. The wound repair result was evaluated by estimation of the percentage of remaining wound area and the amount of wound bacteria under the scar, followed by observation of pathological changes. Inflammatory reactions of the wounds were assessed accordingly. Results The amount of bacteria under the scar was less in group T than in the other two groups (P<0.05). It was also found that the wound healing process was faster in group T than in group C and group N. Pathological observation showed that the inflammatory reaction in group T was also significantly milder than in the other two groups. Conclusion BPI-BD3 gene-modified mesenchymal stem cells may enhance wound repair by controlling infection and promoting tissue regeneration, thus it may be promising in clinical application.

Evaluation of effect of red ginseng on ovariectomy-induced bone loss in C3H/HeN mice / 동물의과학연구지

Panax ginseng, also known as Korean ginseng, has long been used as a broad tonic in Oriental medicine to augment vitality, health, and longevity, particularly in older people. This study investigated the effects of Korean red ginseng (RG) on bone loss in ovariectomized (OVX) mice. C3H/HeN mice (10-weeks-old) were divided into sham and OVX groups. OVX mice were treated with vehicle, 17beta-estradiol (E2), RG (oral administration, 250 mg/kg/day), or RG (intraperitoneal administration, 50 mg/kg/every other day) for 6 weeks. Serum E2 concentration and alkaline phosphatase (ALP) activity were measured. Tibiae were analyzed using microcomputed tomography. Biomechanical properties and osteoclast surface level were measured. There was no significant difference in the degree of grip strength, body weight, uterine weight, mechanical property, tibiae length, or tibiae weight between the OVX and RG-treated groups. Compared with the OVX group, the serum ALP level was significantly lower in the RG-treated groups. Serum E2 levels and osteoclast surface levels did not change between the OVX and RG-treated groups. RG could not preserve trabecular bone volume, trabecular bone number, trabecular separation, trabecular thickness, structure model index, or bone mineral density of the proximal tibiae metaphysic. In conclusion, there was no definite effect of RG on OVX-induced bone loss in C3H/HeN mice.

O papel do flúor sobre linhagens de células osteoblásticas de camundongos com densidades ósseas distintas: estudos em MC3T3-E1, C3H/HeJ e C57BL/6J / The role of fluoride on different osteoblastic cell line from different mice strains with distinct bone density: Study on MC3T3-E1, C3H/HeJ and C57BL/6J

Sabe-se que o flúor causa alterações químicas e celulares no osso, dependendo da dosagem e do tempo de exposição a esse elemento. Já foi demonstrado que alterações clínicas importantes, como a osteoporose, osteopetrose e esclerose óssea, podem ser geradas pela ingestão de flúor. Sabe-se que o flúor pode guiar os osteoblastos ao crescimento, sob doses e tempos de exposição específicos. A sensibilidade dos tecidos mineralizados ao fluoreto responde a mecanismos genéticos inerentes a cada população. Dentro de uma mesma espécie, diferentes linhagens apresentam um comportamento distinto frente a esse fármaco. Durante várias décadas, os estudos com fluoretos resumiram-se ao fluoreto de sódio, amplamente utilizado na prevenção das cáries dentárias. Recentemente, outras fontes de fluoretos como TiF4 e SnF2 têm sido buscadas, com intuito de potencializar as vantagens desse elemento. Porém não existem estudos ainda sobre o efeito do AlF3 sobre o tecido ósseo até a presente data. Desse modo, achamos oportuno investigar seu papel, quando administrado a células ósseas e para tal, investigamos a viabilidade e padrão de atividade das fosfatases desse sal (AlF3) quando administrado às células da linhagem pré-osteblástica MC3T3, em comparação ao NaF. Além disso, sabendo que diferentes células apresentam comportamento distinto ao tratamento com fluoretos, estudamos o papel do NaF quando administrado a duas linhagens celulares de camundongos com densidades ósseas distintas e com diferentes susceptibilidades ao fluoreto: C3H/HeJ e C57BL/6J (C3H e C57, alta e baixa densidades ósseas, respectivamente). As células também foram avaliadas quanto à viabilidade celular e ao perfil das fosfatases. Todos os resultados foram submetidos à Análise de Variância a três critérios e nos permitiu identificar que entre os sais de fluoreto, apenas o NaF promoveu alterações na viabilidade celular das células MC3T3. O AlF3 não exerceu influência na atividade da fosfatase alcalina...

It is well known that fluoride causes chemical and cellular alteration on bone tissue, depending on the dosage and time of exposure to this element. Several studies have been conducted to show that some bone diseases, such as osteoporosis, osteopetrosis and esclerosis, can occur as a consequence of the excessive fluoride uptake. Fluoride can lead osteoblast to proliferate, under certain conditions as time and dosage. The same way, excessive dosages of fluoride can alter protein activity in osteoblasts, by changing the expression of retated genes. Sensitivity of mineralized tissues to fluoride depends on a genetic background inherent to each population. Within the same specie, different lineages present distint behavior to the ingestion of this íon. Over several decades, many researches focused on the study of NaF as the only source of fluoride, once it is wide spread used to the prevention of dental caries. More recently, different sources of fluoride have been assessed, like TiF4 e SnF23 did not exibit influence over on alkaline phosphatase when pre-osteoblast cells are considered, but the effect of this fluoride salt on the activity of total and tartarate resistant acid phosphatases ocurrs on a dicotomic way. When we consider a mixed cell population, collected from calvária as for the C3H and C57 assays, NaF did interfered as with the proliferation rate as phosphatase activities on a very distinct fashion between the two lineages. Thus, it is possible to conclude that NaF is able to exert variable effects in different cell types and enzyme activities; on the other hand, AlF3 effects are more specific and surrounded in pre-osteblastic cells.

O papel do flúor sobre linhagens de células osteoblásticas de camundongos com densidades ósseas distintas: estudos em MC3T3-E1, C3H/HeJ e C57BL/6J / The role of fluoride on different osteoblastic cell line from different mice strains with distinct bone density: study on MC3T3-E1, C3H/HeJ and C57BL/6J

Sabe-se que o flúor causa alterações químicas e celulares no osso, dependendo da dosagem e do tempo de exposição a esse elemento. Já foi demonstrado que alterações clínicas importantes, como a osteoporose, osteopetrose e esclerose óssea, podem ser geradas pela ingestão de flúor. Sabe-se que o flúor pode guiar os osteoblastos ao crescimento, sob doses e tempos de exposição específicos. A sensibilidade dos tecidos mineralizados ao fluoreto responde a mecanismos genéticos inerentes a cada população. Dentro de uma mesma espécie, diferentes linhagens apresentam um comportamento distinto frente a esse fármaco. Durante várias décadas, os estudos com fluoretos resumiram-se ao fluoreto de sódio, amplamente utilizado na prevenção das cáries dentárias. Recentemente, outras fontes de fluoretos como TiF4 e SnF2 têm sido buscadas, com intuito de potencializar as vantagens desse elemento. Porém não existem estudos ainda sobre o efeito do AlF3 sobre o tecido ósseo até a presente data. Desse modo, achamos oportuno investigar seu papel, quando administrado a células ósseas e para tal, investigamos a viabilidade e padrão de atividade das fosfatases desse sal (AlF3) quando administrado às células da linhagem pré-osteblástica MC3T3, em comparação ao NaF. Além disso, sabendo que diferentes células apresentam comportamento distinto ao tratamento com fluoretos, estudamos o papel do NaF quando administrado a duas linhagens celulares de camundongos com densidades ósseas distintas e com diferentes susceptibilidades ao fluoreto: C3H/HeJ e C57BL/6J (C3H e C57, alta e baixa densidades ósseas, respectivamente). As células também foram avaliadas quanto à viabilidade celular e ao perfil das fosfatases. Todos os resultados foram submetidos à Análise de Variância a três critérios e nos permitiu identificar que entre os sais de fluoreto, apenas o NaF promoveu alterações na viabilidade celular das células MC3T3. O AlF3 não exerceu influência na atividade da fosfatase alcalina...

It is well known that fluoride causes chemical and cellular alteration on bone tissue, depending on the dosage and time of exposure to this element. Several studies have been conducted to show that some bone diseases, such as osteoporosis, osteopetrosis and esclerosis, can occur as a consequence of the excessive fluoride uptake. Fluoride can lead osteoblast to proliferate, under certain conditions as time and dosage. The same way, excessive dosages of fluoride can alter protein activity in osteoblasts, by changing the expression of retated genes. Sensitivity of mineralized tissues to fluoride depends on a genetic background inherent to each population. Within the same specie, different lineages present distint behavior to the ingestion of this íon. Over several decades, many researches focused on the study of NaF as the only source of fluoride, once it is wide spread used to the prevention of dental caries. More recently, different sources of fluoride have been assessed, like TiF4 e SnF23 did not exibit influence over on alkaline phosphatase when pre-osteoblast cells are considered, but the effect of this fluoride salt on the activity of total and tartarate resistant acid phosphatases ocurrs on a dicotomic way. When we consider a mixed cell population, collected from calvária as for the C3H and C57 assays, NaF did interfered as with the proliferation rate as phosphatase activities on a very distinct fashion between the two lineages. Thus, it is possible to conclude that NaF is able to exert variable effects in different cell types and enzyme activities; on the other hand, AlF3 effects are more specific and surrounded in pre-osteblastic cells.

Protective effect of Minocycline on photoreceptor cell in retinal pigmentary degeneration mice / 中华眼底病杂志

Objective To observe the effect of Minocycline on RP process of retinal pigmentarydegeneration rd mice[C3H/HeN(Pde6brd-/rd-)]. Methods 40 rd mice were divided into ten groupsrandomly:5 experimental groups and 5 control groups,4 rd mice in each group.The experimental groupreceived intraperitoneal injection of minocycline 22.5mg/kg while the control group received saline 10ml/k2 every day from the posmatal day 1(P1).Mice were sacrificed at P1,P7,P14,P21 and P28respectively.Eyeballs were enucleated to carry out histology observation and apoptosis cell detection.Meanwhile,to statistically analyze the number of retinal photoreceptor cells,the thickness of outer nuclearlayer(ONL)and the number of apoptosis cells. Results (1)Photoreceptor cell began to apoptosis onP7,peaked on P14,and totally disappeared on P28.(2)No statistically significant differences were foundof the number of photoreceptor cells and the thickness of ONL on P7 between the experimental group andthe control group.(3)The number of photoreceptor cells and the thickness of ONL in the experimentalwere more than that in the control group at P14,P21,P28 respectively,the differences are statisticallysignificant(P＜0.05).(4)The apoptotic cells on ONL were less in the experimental group than that in thecontrol group on P7 and P14 respectively,the difference are statistically significant(P＜0.05).Conclusions Minocycline appears to protect photoreceptor cell from apoptosis in the early stage of theretinal degeneration mice,but it may not completely prevent RP from occurrence.

A Primary Study of the Subgroups of T Lymphocytes in MHV-3 Induced Chronic Viral Hepatitis / 中国病毒学

To study the contribution of T cell subsets in the pathogenesis of Murine hepatitis virus Type3 (MHV-3) induced chronic viral hepatitis in C3H/Hej mice, ninety C3H/Hej mice were chosen to individually receive 10 plaque forming units (PFU) of MHV-3 intraperitoneally. The changes of virus titer and pathology in liver tissue were examined by standard plaque assay and by the hematoxylin/eosin (HE) staining method from 2 days post MHV-3 infection. The ratios of T cell subsets including CD3+CD4+CD8-, CD3+CD4-CD8+, CD3+CD4-CD8-, CD3+CD4+CD25+, CD3+CD4+CD25- and CD3+CD4-CD25+ T lymphocyte of total T lymphocytes in blood, spleen and liver were examined at 0, 2, 4, 6,8, 10, 12, 15, 20, 25, 30, 40 days post MHV-3 infection by flow cytosorting. We observed that the virus titer raised and showed persistent virus duplications and inflammatory changes in the livers of C3H/Hej mice from 2 days post MHV-3 infection. The double negative T cell (DN Treg cell) and CD4+CD25+ T cell ratios increased significantly from 2 days post MHV-3 infection in C3H/Hej mice, and CD3+CD4+CD8-, CD3+CD4-CD8+, CD3+CD4+CD25- and CD3+CD4-CD25+ T cell ratios decreased accordingly. In conclusion, the changes of virus titer and pathology in the livers of C3H/Hej mice post MHV-3 suggest their contribution to viral persistence. Further characterizations of DN Treg cells are that infection indicates that MHV-3 could induce the chronic inflammation in livers of C3H/Hej mice.The increase of the DN Treg cell and CD4+CD25+ T cell ratios in C3H/Hej mice post MHV-3 infection suggests that DN Treg cells and CD4+CD25+ T cells may both have important suppressive immunomodulation functions in the development of chronic viral hepatitis and have important roles in the virus persistent infection. Further characterizations of DNT cell and CD4+CD25+ T cell are under investigation.

EFFECTS OF PROSTAGLANDIN E2 PRODUCTION ON LPS-INDUCED REDUCTION IN WHEEL-RUNNING ACTIVITY IN MICE / 体力科学

To determine whether lipopolysaccharide (LPS)-induced prostaglandin (PG) E2 production is responsible for reduced spontaneous physical activity, we measured LPS ( 1 mg/kg, i. v.)-induced changes in voluntary wheel-running activity for 24 hours in both C3H/HeJ (LPS unresponsive due to a mutation in the <i>tlr4</i> gene) and C3H/HeN (LPS response) mice. We also examined the effect of <i>tlr4</i>-gene mutation on LPS-induced PGE2 production using peritoneal macrophages from the C3H/HeJ and C3H/HeN mice. In addition, the voluntary wheel-running activity of the C3H/HeN mice, which were injected with the PGE2 inhibitor indomethacin (IM ; 0-20 mg/kg, i. p.) 30 min before injection with or without LPS ( 1 mg/kg), was monitored for 24 hours. Wheel-running activity in the C3H/HeJ mice was maintained in spite of LPS injection, but the activity in the C3H/HeN mice was significantly reduced by LPS injection. <i>In vitro</i> experiment showed peritoneal macrophage PGE2 production to be lower in the C3H/HeJ mice than that in the C3H/HeN mice. IM partially, but significantly, attenuated the LPS-induced reduction in wheel-running activity in the C3H/HeN mice. Our results suggest that the transient reduction in physical activity after LPS injection is partially mediated by LPS-induced PGE2 production, and that other factors also play a role.

Experimental study of establishing mouse model for scleroderma / 中华风湿病学杂志

Objective To establish a mouse model for scleroderma.Methods Forteen BALB C and 14 C3H female mice were averagely divided into model 1 and controls.Daily 0 1 ml BLM at a concentration of 200 ?g/ml was injected intracutaneusly into the backs of model 1 mice for 3 weeks,and 0 1 ml solution of PBS were injected intracutaneusly into the backs of control mice for 3 weeks.Observing the histological change of skin and lungs was made and measuring the thickness of dermis was performed with a medical analysis system of the color picture,determined the collagenic quantity was done with photoelectric colorimetry,and calculating the immunohistochemical index of collagen type Ⅰ and Ⅲ and transforming growth factor ? 1 (TGF ? 1) in the skin lesions from the mouse model and control was done.SPSS was used to finish the statistical analysis of the detective value from model 1 and controls.Results In the skin of model mice,the thickness of dermis markedly thickened ( P

The influence of BMP_9 in myocardiocytes differentiation of stem cell in vitro / 重庆医科大学学报

Objective:ToexploreinfluenceofBMP9inmyocardiocytesdifferentiationof stem cell Invitro.Methods:TheC3H10T1/2stem cell were transfected with 2.67 ml/L pAdEasy-BMP9 Ad-GFP plasmid for 1 week and 2 weeks .The specific transcription factors of cardiomyocytes were detected in stem cells during differentiation with plasmid transfection by RT-PCR and the specific proteins in cardiomy-ocytes were detected by laser confocal microsopy after 1 week;the change of cells ultramicro structure were detected by electron micro-scope after 2 weeks.Results:Cells volume are increscent obviously,cells trend become unanimous,the connection between cells are compact,refractivity of cells enhance conspicuously after induction for 1 week.The expression of NKx 2.5,GATA-4,MEF2C could be de-tected in C3H10T1/2 stem cells with pAdEasy-BMP9 Ad-GFP plasmid tranfection.Cx43 and cTnT can be detected with plasmid tranfec-tion.Cardiomyocytes ultramicro structure can be detected after induction for 2 weeks.Conclusion:BMP9 makes a very important influ-ence in C3H10T1/2 stem cells targeted differentiation into cardiomyocytes。

Promoting Effect of a Mixture of 8 Herbal Extracts (SPELA 707) on Hair Growth

In Korean folk medicine, several herbs, Glycyrrhizae Radix, Persicae Semen, Salviae Radix, Angelicae Gigantis Radix, Zanthoxyli Fructus, Ginseng Radix Alba, Cnidii Rhizoma, and Carthami Flos, are known to enhance blood circulation and have wound healing or anti-inflammatory effects. These pharmacological actions prompted us to investigate whether these herbs might stimulate hair growth. Thus, using a mixture of their extracts called SPELA 707, we investigated their effects and found that SPELA 707 possessed significant hair cycle converting activity from the telogen phase to the anagen phase in C3H mice. Furthermore, we found that SPELA 707 enhanced the hair density in subjects with hair loss and also promoted the conversion of hair into the anagen phase in subjects with androgenetic alopecia. In addition, hair growth promotion effect of SPELA 707 occurred through inhibition of steroid 5 alpha-reductase activity, which is known to block hair growth. Taken together, these results suggest that SPELA 707 has a potential to be used for the treatment of hair loss.