Mechanism Of Long The Baicalensis Grass Sprays On Isolated Rat Alveolar Type ¢ò Epithelial Cells

Alveolar type Ⅱ cells(AT Ⅱs)are important structure cells that line the alveolar surface.They act as the stem cell for the alveolar epithelium to proliferate and further differentiate intotype I cells, and play a critical role in the repair of lung injuries。Purposes: The purposes of this study are to make a ALI model of cultured alveolar type Ⅱ cells invitro, it's induced by LPS(lipopolysaccharide,LPS). Under this model we'll investigate thealteration of alveolar type Ⅱ cells' apoptosis,secretory function. At the same time we use atraditional medicine intervente the injury process to observe the medicine'role.Methods: In the first part,alveolar type Ⅱ cells were isolated by Dobbs's method what had beenimproved, the cells puritied by adhered to IgG coated dishes and identified with a tannic acidstain and AKP stain. The cellular ultra-structure in alveolar type Ⅱ cells were observed bythese two method. In the second part, We add different doses of LPS from 10ug/ml to 100ug/ml in culturemedium to stimulate alveolar type Ⅱ cells for 2 hours. the concentrations of TNF- and IL-8 inthe culture media were determined based on ELISA . Fluorescence- quantitative-PCR techniquewas performed to quantitate the levels of SP-A,SP-B and AQP-1mRNA in alveolar type Ⅱcells.Results: 1. Isolating, puritying and identifying alveolar type Ⅱ cells of rats: We get the 109 cellsbefore purification and 107 cells after it. The trypan blue stain shows that the cells' activity overthe 96 percent. Identified with a tannic acid stain and AKP stain, before purification thepuritying is 74.5±13.8%, after is 90.4±2.6%. 2. It was shown that when the alveolar type Ⅱ cells were exposed to different doses ofLPS from 10ug/ml to 100ug/ml for 2 hours in culture medium, LPS could result in the necrosisand apoptosis of cultured alveolar type Ⅱ cells in this model in vitro. 3. It was shown that when the alveolar type Ⅱ cells were exposed to different doses ofLPS from 10ug/ml to 100ug/ml for 2 hours in culture medium , LPS could stimulate secretion ofTNF-α of alveolar type Ⅱ cells significantly increased as compared with control group. Afteruse the medicine the increase will be inhibited obviously. The secretion of IL-8 have not asignificantly increase. 4. The expression of SP-A,B and AQP-1 in LPS injured type Ⅱcells was significantlydecreased as compared with control group,P<0.001。After use the traditional Chinesemedicine this effect could be inhibited, P<0.001。Conclusions:- 4 - 龙芩草喷雾剂对大鼠离体肺泡Ⅱ型上皮细胞的作用机理研究 1. Isolating, puritying and identifying alveolar type Ⅱ cells of rats: fitting theconcentration of digestive enzyme and digestion time could rise the cellular vitality. Adhered toIgG coated dishes after isolating provided more high cell purity than before. It could be foundthe lamellar bodies with a tannic acid stain andAKP stain. 2. LPS could injured alveolar type Ⅱ cells directly in vitro. The manifestation was include:it could result in the necrosis and apoptosis of cultured alveolar type Ⅱ cells, make thesecretion of TNF-αof alveolar type Ⅱ cells significantly increased, and the expression ofSP-A,B andAQP-1 was significantly decreased. 4. After use the traditional medicine these injury are all inhibited. It is implicated that thismedicine might directly inhibit LPS induced injury of alveolar type Ⅱ in vitro.