| Bovine mastitis is one of the most important diseases of dairy cow throughout the world. It is also a main cause of economic losses to the dairy industry. The most frequent infective agents causing mastitis in dairy cattle is Escherichia coli, one of the Gram-negative bacteria. LPS is an important structural component of the outer membrane of Gram-negative bacteria. Toll-like receptor4(TLR4), a pathogens receptor, plays a key role in triggering the innate immune response. LPS/TLR4signaling among the bovine mastitis has been intensively studied in the past few years. TLR4recognizes the conserved parts of LPS and activates a signaling cascade that leads to activation the transcription factor, nuclear factor κ B or NF-κB, increase the expression of pro-inflammatory cytokines. To analyze the expression of TLR4and TNF-α in mammary glands, mice mammary glands were stimulated by LPS in vitro and in vivo, and the results were discussed.1The expression of TLR4and TNF-α mRNA in mice mammary glands after inoculation with LPSForty ICR female mice were divided into test group and control group after conception. On days10to11of lactation, two abdominal mammary glands (L4and R4) of mice were infused with O.1mL of either LPS solution (20μg/mL) or saline solution through needle. On the1.5h、3h、6h、12h after injection the mammary glands were collected, one-half of each mammary gland was performed HE staining for histological analysis; a portion of the remaining tissue was used for RNA extraction. TLR4and TNF-α mRNA expression was analysised after using RT-PCR. Result:Few quantity of inflammatory cells infiltrated in the mammary glands at1.5h after intramammary infusion of LPS; Large quantity of inflammatory cells infiltrated in the mammary glands and normal alveolar architecture was lost at6h,12h postinjection. The mRNA levels of TLR4and TNF-α increased rapidly after stimulation with LPS, the expression of TLR4mRNA at6h after intramammary infusion was extremely significantly higher than that of control (p<0.01); TNF-α mRNA expression in test group was also extremely significantly higher than that of control (p<0.01). 2Establishment of a cell culture method of mammary epithelial mammary cellTwo abdominal mammary glands from pregnant (14-19days) ICR mice were removed for primary cell culture. The tissue was digested with collagenase to obtain separate cells. Observe the morphology of mammary epithelial cells, using MTT assay to draw the cellular growth curve, make sure the exponential growth phase of mammary epithelial cells. Most of the cell clumps were adherenced at24h after inoculation; cells derived from the clumps cultured48h, the volume of mammary cells increased;72h after cell inoculation, cell displayed a typical cuboidal epithelial-like morphology and formed monolayers with the cells in close contact with each other; at96h80%-90%cells confluent;120h cells failed to grow. From the growth curve good viability of mammary epithelial cells could be seen, and was concordant to normal cells tendency.3The expression of TLR4and TNF-α mRNA in mammary epithelial cells after stimulating with LPSEstablishment of the primary mammary epithelia cell cultures were divided into test groups and control group. Cells were stimulated with2mL LPS (1μg/mL), and2mL DMEM-F12as control. After1.5h、3h、6h、12h stimulation, the medium was removed and the cells were homogenized for RNA extraction. RT-PCR and analysis the expression of TLR4and TNF-α mRNA were carried out. The experiments were repeated three times. Result showed the mRNA levels of TLR4and TNF-α increased rapidly after stimulation with LPS, the expression of TLR4mRNA at1.5h,3h,6h after intramammary infusion was significantly higher than that of control (p<0.05); TNF-α mRNA expression at1.5h was significantly higher than that of control (p<0.05). |
PDF Full Text Request