Effect of Flos Buddlejae Granules on Apoptosis Factors Bax,Bcl-2,Fas and FasL in Lacrimal Gland Cells of Castrated Male Rabbits

Objective To observe the effect of different concentration Flos Buddlejae(Mi Meng Hua,密蒙花)granules on apoptosis factors of lacrimal gland cells of castrated male rabbits,and study the treatment effects of Flos Buddlejae(Mi Meng Hua,密蒙花)granules in the dry eye model of castrated male rabbits.Methods Flos Buddlejae(Mi Meng Hua,密蒙花)raw material was made into granules.Thirty healthy adult New Zealand rabbits were randomly divided into five groups,six rabbits in each group.Group A:blank group,Group B:model group,Group C:Flos Buddlejae(Mi Meng Hua,密蒙花)granule group,Group D:placebo group,Group E:testosterone group.Except for group A,all rabbits underwent removal of bilateral testis and epididymis.Rabbits in group C were administered Flos Buddlejae(Mi Meng Hua,密蒙花)granules(100 mg/kg),three times per day;rabbits in group D were administered normal saline,three times per day.Rabbits in group E were injected with testosterone propionate(0.5 mL/kg)in the thigh muscle,every 3 days.All rabbits were tested by Schirmer I test(SIT)and tear film break-up time(BUT)before operation and 4 weeks after operation.After 4 weeks,all rabbits were sacrificed by air embolism and clipping of the lacrimal gland.Apoptosis factors,including Bax,Bcl-2,Fas and FasL of lacrimal gland cells were characterized by immunohistochemistry,and resulting data were statistically analyzed.Results(1)Comparison of SIT and BUT before and after operation:There were statistically significant differences between groups B and D(P<0.01),but not among other groups(P>0.05).(2)Comparison of apoptosis factors Bax,Bcl-2 Fas and FasL:In a comparison of groups B and D,there was no statistically significant difference after operation(P>0.05).In a comparison of the other groups,there were statistically significant differences(P<0.01).In comparisons among A,C and E groups,there were no statistically significant differences(P>0.05).Conclusion Compared with androgen,Flos Buddlejae(Mi Meng Hua,密蒙花)granules caused similar but slightly weaker depression of Bax,Fas and FasL,and increased expression of Bcl-2.

The effects of thyroid-stimulating hormone,estradiol and prolactin on sodium/iodide symporter mRNA expression in mouse lactating mammary gland cells under different iodine levels

Objective The present study investigated the sodium/iodide symporter mRNA expression in mouse lactating mammary gland cells under different iodine levels and the effects of thyroid-stimulating hormone(TSH),estradiol(E2)and prolactin(PRL)on NIS mRNA expression in mouse lactating mammary gland cells.

Influence on Cellular Signal Transduction Pathway in Dairy Cow Mammary Gland Epithelial Cells by Galactopoietic Compound Isolated from Vaccariae segetalis

The galactopoietic mechanism of Vaccaria segetalis is still unknown. Understanding dibutyl phthalate （DBP） separated from Vaccaria segetalis on the expression of lactation signal transduction genes of mammary gland epithelial cells, including prlr, erα, akt1, socs2, pparγ and elf5, will be helpful to reveal the molecular mechanism. Western blot and qRT- PCR were used to study the change of prlr, erα, akt, socs2, pparγ, and elf5 expression at mRNA and protein level. Co- localization expression of prolactin receptor （PRLR） and estrogen receptor α （ERα） was observed by immunofluorescence; the expression changes of miRNAs （21, 125b, 143, and 195） and the secretion of β-casein and lactose were detected by qRT-PCR and RP-HPLC. The results showed that Vaccaria segetalis active compound had similar fuctions as estrogen and/or prolactin （PRL） in dairy cow mammary gland epithelial cells （DCMECs）, increased the expressions of prlr, erα, akt1, and elf5 genes, while repressed pparγ expressions. DBP promoted socs2 mRNA expression, but its protein expressions were repressed. Furthermore, both DBP and PRL could repress the expressions of miRNA-125b, miRNA-143 and miRNA- 195 in DCMECs. DBP could repress the expression of miRNA-21, while the influence of PRL on miRNA-21 was not certain. DBP could promote the lactation ability of DCMECs by regulating the ER and PRLR cellular signal transduction pathway.

Metabolic Regulation of Mammary Gland Epithelial Cells of Dairy Cow by Galactopoietic Compound Isolated from Vaccariae segetalis

In previous experiment, we isolated a compound dibutyl phthalate （DBP） from Vaccaria segetalis which had galactopoietic function on mammary gland epithelial cells of dairy cow （DCMECs）. In this experiment, we ascertained the metabolic regulation function of DBP on DCMECs. Many genes related to lactation including Stat5, AMPK, b-casein, Glut1, SREBP-1, PEPCK, and ACC were detected by real-time PCR. Furthermore, Stat5 and AMPK were detected by Western blot and immunofluorescence co-localization, respectively. The results showed that DBP stimulates the expression of Stat5 and p-Stat5, thus activates Stat5 cell signal transduction pathway and stimulates b-casein synthesis. DBP also raises the activities of Glut1 and AMPK to stimulate glucose uptake and glycometabolism and activates the expression of AMPK downstream target genes PEPCK and ACC and expression of SREBP-1 to stimulate milk fat synthesis. In addition, the activities of HK, G-6-PDH, ICDH, ATPase, and energy charges were stimulated by DBP to increase the energy metabolism level of DCMECs. The results showed DBP stimulates energy metabolism related to galactopoietic function in DCMECs.

Construction of Antibacterial Peptide CecropinB Eukaryotic Recombinant Vector and Its Expression in Dairy Goat Mammary Gland Epithelial Cells

To investigate the expression of antibacterial peptide CecropinB cDNA in dairy goat mammary gland epithelial cells,the CecropinB gene was cloned and was inserted into a eukaryotic vector pECFP-C1 to construct the recombinant plasmid pECFP-B by genetic engineering technique.Recombinant plasmid pECFP-B was transfected into dairy goat mammary gland epithelial to detect the bactericidal activity of CecropinB.The expression of CecropinB was also detected.The result of RT-PCR demonstrated CecropinB gene was expressed in transfected cells.CecropinB recombinant plasmid DNA was injected into udders and CecropinB was expressed in mammary gland,exhibiting bactericidal activity to Staphylococcus aureus in vivo experiments.

Comparison on In-Vitro Culture Methods of Yak Endometrial Gland Epithelial Cells

[ Objective] To develop in-vitro culture methods of yak endometrial gland epithelial cells. [Method] The gland epithelial cells were isolated from yak endometrium by explant culture method and digestion culture method, respectively. [ Result] In the first method, the cells isolated from the endometrium explant could merge into monolayer after 8-d culture, and they could be purified by gradation digestion with trypsin. In the second method, the endometrium explant were first digested by collagenase II by incubation at 37℃ for 2.5 h and then further digested in fresh 2 g/L colla- genase II for another 2.5 h. The cell suspension was leached through 74 pm filter and centrifuged at 400 r/min for 5 min. Then the cell pellet was re-suspended, followed by natural sedimentation to collect purified gland epithelial cells. The isolated cells were cytokeratin-positive as detected by immunocytochemical staining, and the positive rate could reach 95%. [Conclusion] The yak endometrial gland epithelial cells can be isolated and purified by both the explant culture method and digestion culture method.

Characterization of dual enzyme resulted from bicistronic expression of two β-glucanases in porcine cells

Many animal feed grains contain high β-glucan in the cell wall. Pigs do not secret β-glucanase to degrade the β-glucan in their feed. The indigestible β-glucan not only blocks the release of nutrients from the grain cell wall, but also increases the digesta viscosity in the gastrointestinal tract of pigs. Therefore, dietary β-glucan significantly inhibits nutrient digestion and absorption in pigs. Transgenic expression of β-glucanase in the digestive tract of pigs may offer a solution to solve this problem. In the current study, four artificial codon-optimized β-glucanases genes was prepared and expressed in porcine cells. Only p Bg A and p Egx showed high activity in transfected pig kidney cells. To improve the p H range and p H stability of β-glucanase, the two β-glucanases, p Bg A and p Egx, were co-expressed in pig kidney cells and salivary gland cells by Linker A3 or 2A peptide. The resulting dual enzymes of p Bg A3 p Eg and p Bg2 Ap Eg showed significantly enlarged p H range and significantly increased p H stability, as compared to parental enzymes. These results provide useful data for future study on increasing the feed digestibility of pigs by transgenic expression of β-glucanase in their salivary glands.

Induced in vitro Expression of Human Lactoferrin in Goat Mammary Gland Epithelial Cell

[Objective]The aim of this study was to explore the technical system of induced expression in vitro of goat mammary gland epithelial cell,and evaluate expression efficiency of mammary gland specific vector and foreign protein at the cell level.[Method]Goat mammary gland epithelial cell transfected by human lactoferrin gene was inducted by culturing in DMEM/F12 medium supplemented with 5 mg/L insulin,5 mg/L prolactin and 1 mg/L hydrocortisone.Supernatant was collected per 6 hours and concentrated.Expression situation of foreign protein were detected by SDS-PAGE and Western blotting.[Result]There was target protein expression in the induced culture medium,which molecular weight was about 42 kD.[Conclusion]The method used in this study can induce goat mammary gland epithelial cell to express foreign gene,it lays a foundation for researching heterologous expression of foreign gene and producing mammary gland bioreactor.

Effect of Different Concentrations of Lipopolysaccharide on Expression of NF-κB and LAP in Mammary Epithelial Cells of Dairy Cow

The paper was to study the effects of different concentrations of lipopelysaccharide （LPS） on expression of nuclear factor Kappa B （NF-κB） and lingual antimicrobial peptide （LAP） gene in mammary epithelial ceils of dairy cow. The mammary epithelial ceils of dairy cow were stimulated by different concentrations （50, 100,200,400 and 800 ng/mL） of LPS. The total RNA of cells was extracted after stimulation for 2, 4, 8, 16, 24, 48 and 72 h, respectively, and the mRNA expression levels of NF-κB P65 and LAP were evaluated by real-time quantitative PCR. The results showed that the expression of NF-κB P65 and LAP mRNA treated with 400 ng/mL LPS for 72 h were the highest compared to the control group （ P 〈0.01 ）. The result confirmed that the expression activity of NF-κB was enhanced in inflammatory effects of inammary epithelial cells induced by LPS, which regulated the expression of defense gene LAP, with certain dose and time effects.

Inhibition of Salidroside on Salivary Gland Adenoid Cystic Carcinoma

To evaluate the role of salidroside on proliferation,apoptosis and invasiveness of salivary gland adenoid cystic carcinoma cells（SACC）,immunocytochemical staining was employed to detect proliferating cell nuclear antigen（PCNA）,caspase 3 and caspase 8 expression in SACC-2 cells.Modified Boyden chamber assay combined with laser confocal microscopy（LSCM） was used to evaluate the invasion and migration abilities of SACC-2 cells at different time point.Immunohistochemistry staining revealed that the expression of PCNA was significantly decreased（P0.01） after salidroside treatment.In contrast,salidroside treatment led to increased caspase 3 and caspase 8 in SACC-2 cells.Cell migration depth and number of cells that penetrated Boyden chamber were also decreased by salidroside.Salidroside potently inhibits the proliferation and simultaneously induces the apoptosis of SACC-2 cells.Migration and invasion of SACC-2 cells are also inhibited.Our data throw light on potential clinical application of salidroside to the patients with SACC.

Galactopoietic Activity of Dibutyl Phthalate Isolated from Vaccaria segetalis

A galactopoietic compound, identified as dibutyl phthalate（DBP）, was isolated from Vaccaria segetalis. The activity of DBP on lactation ability of dairy cow mammary gland epithelial cells（DCMECs） cultured in vitro and dairy cow was evaluated. Results showed that DBP could promote cell viability, proliferation ability, lactose and β-casein secretion of DCMECs, which could also raise the milk yields of dairy cows significantly.

3' Noncoding Region Construction of GHR Gene-luciferase Report Vector and Valuation

To analyze miR-139 target sites in 3＇ UTR of GHR gene in dairy cow mammary gland, a GHR 3＇ UTR- luciferase reporter vector was constructed and the effect of miRNA on its activity was evaluated in dairy cow mammary gland epithelial cells （DCMECs）. The miR-139 targeting GHR 3＇ UTR was predicted by Target Scan 5.1 software, 3＇ UTR fragment of GHR was amplified by PCR from RNA of DCMECs. PCR products were cloned into Spe Ⅰ/Hind Ⅱ modified pMIR-Report vector. The luciferase reporter vector and miRNA eukaryotic expression vector were transferred into DCMECs using lipofectamine 2000 transfection reagent. The dualluciferase reporter assay system was used to quantitiate the reporter activity. The results showed that a 107 bp 3＇ UTR fragment of GHR gene was successfully cloned into the pMIR-Report vector, which authenticated by Spe Ⅰ/Hind Ⅲ digestion and DNA sequencing. The luciferase activity of reporter construction treated with miR-139 decreased 20.87% compared with the control group. It was concluded that the GHR3＇ UTR-luciferase reporter vector had been successfully constructed. The luciferase activity of the reporter could be suppressed by miR- 139.

TNF-α suppresses sweat gland differentiation of MSCs by reducing FTO-mediated m^6 A-demethylation of Nanog mRNA

An effect of inhibition of tumor necrosis factor-α(TNF-α)on differentiation of mesenchymal stromal cells(MSCs)has been demonstrated,but the exact mechanisms that govern MSCs differentiation remain to be further elucidated.Here,we show that TNF-αinhibits the differentiation of MSCs to sweat glands in a specific sweat gland-inducing environment,accompanied with reduced expression of Nanog,a core pluripotency factor.We elucidated that fat mass and obesity-associated protein(FTO)-mediated m^6 A demethylation is involved in the regulation of MSCs differentiation potential.Exposure of MSCs to TNF-αreduced expression of FTO,which demethylated Nanog m RNA.Reduced expression of FTO increased Nanog m RNA methylation,decreased Nanog m RNA and protein expression,and significantly inhibited MSCs capacity for differentiation to sweat gland cells.Our finding is the first to elucidate the functional importance of m^6 A modification in MSCs,providing new insights that the microenvironment can regulate the multipotency of MSCs at the post-transcriptional level.Moreover,to maintain differentiation capacity of MSCs by regulating m^6 A modification suggested a novel potential therapeutic target for stem cellmediated regenerative medicine.