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
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摘要
肺泡Ⅱ型上皮细胞是肺内的重要结构细胞,它作为肺泡干细胞能增殖分化为肺泡Ⅰ
型上皮细胞,在肺损伤后肺泡上皮的修复与更新过程中有重要意义。
目的:本实验的目的是用脂多糖(lipopolysaccharide,LPS)复制急性肺损伤时的
肺泡Ⅱ型上皮细胞体外损伤模型,在独立环境下观察肺泡Ⅱ型上皮细胞在增殖和分泌功
能方面的损伤情况。同时应用已在临床证明能辅助治疗和预防感染性急性肺损伤的龙芩
草喷雾剂进行干预,探讨此复方的作用机理。
方法:
第一部分:按照Dobbs 等提供的方法并适当改进分离大鼠肺泡Ⅱ型上皮细胞,用IgG
免疫粘附的方法纯化分离的细胞,细胞鉴定采取鞣酸染色和AKP 方法。
第二部分:应用体外培养的大鼠肺泡Ⅱ型上皮细胞,观察脂多糖对其分泌功能及
SP-A、SP-B 及 AQP1mRNA 表达的影响,同时进行龙芩草喷雾剂干预观察药物的作用。通
过酶联免疫吸附法测定细胞培养上清液中TNF-α和IL-8 水平变化,运用FQ-PCR 方法观
察肺泡Ⅱ型上皮细胞中SP-A、SP-B 和AQP1mRNA 表达的改变。
结果:
1.大鼠肺泡Ⅱ型上皮细胞分离、纯化和鉴定:通过计数,纯化前可得约 109个/只数
量的细胞,IgG 免疫粘附纯化后约为 107个/只。台盼兰染色细胞活力为 96%以上。通过
鞣酸染色及 AKP 染色鉴定大鼠肺泡Ⅱ型上皮细胞并进行细胞纯度判断,未纯化时,纯度
达74.5±13.8%,纯化后可提高至90.4±2.6%;
2.以 10ug/ml、50ug/ml、100ug/ml 为 LPS 终浓度刺激培养的肺泡Ⅱ型上皮细胞 2
小时,LPS 可导致体外培养肺泡Ⅱ型上皮细胞损伤,表现为肺泡Ⅱ型上皮细胞坏死和凋
亡发生率增高;
3.LPS 损伤后的肺泡Ⅱ型上皮细胞培养上清液中 TNF-α含量明显增加,加药后可抑
制TNF-α的异常增加;IL-8 含量略有增加,但没有明显差异;
4.LPS 损伤后中 SP-A、SP-B 和 AQP-1mRNA 表达明显降低 P<0.001,使用龙芩草喷雾
剂干预后SP-A 和AQP-1 表达回升P<0.001。
结论:
1.大鼠肺泡Ⅱ型上皮细胞的分离、纯化和鉴定:适当的消化酶浓度、时间可提高分
离细胞的活力,细胞分离后用IgG 免疫粘附方法纯化可使纯度提高15%,鞣酸染色和AKP
鉴定可见Ⅱ型细胞上的特征性板层小体。
2.脂多糖单独作用于体外培养的肺泡Ⅱ型上皮细胞可直接损伤细胞,表现为抑制其
增殖;刺激其分泌炎症因子TNF-α及IL-8 增加;使SP-A、SP-B 和AQP-1mRNA 的表达明
显下降,抑制肺泡Ⅱ型上皮细胞分泌表面活性蛋白和水通道蛋白的功能。
3.龙芩草喷雾剂干预后这些损伤情况均得到明显改善,提示此龙芩草喷雾剂在临床
- 2 - 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
上对急性肺损伤的治疗有效,其对肺泡Ⅱ型上皮细胞的保护作用是机理之一。
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 into
type 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 in
vitro, it’s induced by LPS(lipopolysaccharide,LPS). Under this model we’ll investigate the
alteration of alveolar type Ⅱ cells’ apoptosis、secretory function. At the same time we use a
traditional 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 been
improved, the cells puritied by adhered to IgG coated dishes and identified with a tannic acid
stain and AKP stain. The cellular ultra-structure in alveolar type Ⅱ cells were observed by
these two method.
In the second part, We add different doses of LPS from 10ug/ml to 100ug/ml in culture
medium to stimulate alveolar type Ⅱ cells for 2 hours. the concentrations of TNF- and IL-8 in
the culture media were determined based on ELISA . Fluorescence- quantitative-PCR technique
was performed to quantitate the levels of SP-A、SP-B and AQP-1mRNA in alveolar type Ⅱ
cells.
Results:
1. Isolating, puritying and identifying alveolar type Ⅱ cells of rats: We get the 109 cells
before purification and 107 cells after it. The trypan blue stain shows that the cells’ activity over
the 96 percent. Identified with a tannic acid stain and AKP stain, before purification the
puritying 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 of
LPS from 10ug/ml to 100ug/ml for 2 hours in culture medium, LPS could result in the necrosis
and 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 of
LPS from 10ug/ml to 100ug/ml for 2 hours in culture medium , LPS could stimulate secretion of
TNF-α of alveolar type Ⅱ cells significantly increased as compared with control group. After
use the medicine the increase will be inhibited obviously. The secretion of IL-8 have not a
significantly increase.
4. The expression of SP-A、B and AQP-1 in LPS injured type Ⅱcells was significantly
decreased as compared with control group,P<0.001。After use the traditional Chinese
medicine 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 the
concentration of digestive enzyme and digestion time could rise the cellular vitality. Adhered to
IgG coated dishes after isolating provided more high cell purity than before. It could be found
the 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 the
secretion of TNF-αof alveolar type Ⅱ cells significantly increased, and the expression of
SP-A、B andAQP-1 was significantly decreased.
4. After use the traditional medicine these injury are all inhibited. It is implicated that this
medicine might directly inhibit LPS induced injury of alveolar type Ⅱ in vitro.
型上皮细胞,在肺损伤后肺泡上皮的修复与更新过程中有重要意义。
目的:本实验的目的是用脂多糖(lipopolysaccharide,LPS)复制急性肺损伤时的
肺泡Ⅱ型上皮细胞体外损伤模型,在独立环境下观察肺泡Ⅱ型上皮细胞在增殖和分泌功
能方面的损伤情况。同时应用已在临床证明能辅助治疗和预防感染性急性肺损伤的龙芩
草喷雾剂进行干预,探讨此复方的作用机理。
方法:
第一部分:按照Dobbs 等提供的方法并适当改进分离大鼠肺泡Ⅱ型上皮细胞,用IgG
免疫粘附的方法纯化分离的细胞,细胞鉴定采取鞣酸染色和AKP 方法。
第二部分:应用体外培养的大鼠肺泡Ⅱ型上皮细胞,观察脂多糖对其分泌功能及
SP-A、SP-B 及 AQP1mRNA 表达的影响,同时进行龙芩草喷雾剂干预观察药物的作用。通
过酶联免疫吸附法测定细胞培养上清液中TNF-α和IL-8 水平变化,运用FQ-PCR 方法观
察肺泡Ⅱ型上皮细胞中SP-A、SP-B 和AQP1mRNA 表达的改变。
结果:
1.大鼠肺泡Ⅱ型上皮细胞分离、纯化和鉴定:通过计数,纯化前可得约 109个/只数
量的细胞,IgG 免疫粘附纯化后约为 107个/只。台盼兰染色细胞活力为 96%以上。通过
鞣酸染色及 AKP 染色鉴定大鼠肺泡Ⅱ型上皮细胞并进行细胞纯度判断,未纯化时,纯度
达74.5±13.8%,纯化后可提高至90.4±2.6%;
2.以 10ug/ml、50ug/ml、100ug/ml 为 LPS 终浓度刺激培养的肺泡Ⅱ型上皮细胞 2
小时,LPS 可导致体外培养肺泡Ⅱ型上皮细胞损伤,表现为肺泡Ⅱ型上皮细胞坏死和凋
亡发生率增高;
3.LPS 损伤后的肺泡Ⅱ型上皮细胞培养上清液中 TNF-α含量明显增加,加药后可抑
制TNF-α的异常增加;IL-8 含量略有增加,但没有明显差异;
4.LPS 损伤后中 SP-A、SP-B 和 AQP-1mRNA 表达明显降低 P<0.001,使用龙芩草喷雾
剂干预后SP-A 和AQP-1 表达回升P<0.001。
结论:
1.大鼠肺泡Ⅱ型上皮细胞的分离、纯化和鉴定:适当的消化酶浓度、时间可提高分
离细胞的活力,细胞分离后用IgG 免疫粘附方法纯化可使纯度提高15%,鞣酸染色和AKP
鉴定可见Ⅱ型细胞上的特征性板层小体。
2.脂多糖单独作用于体外培养的肺泡Ⅱ型上皮细胞可直接损伤细胞,表现为抑制其
增殖;刺激其分泌炎症因子TNF-α及IL-8 增加;使SP-A、SP-B 和AQP-1mRNA 的表达明
显下降,抑制肺泡Ⅱ型上皮细胞分泌表面活性蛋白和水通道蛋白的功能。
3.龙芩草喷雾剂干预后这些损伤情况均得到明显改善,提示此龙芩草喷雾剂在临床
- 2 - 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
上对急性肺损伤的治疗有效,其对肺泡Ⅱ型上皮细胞的保护作用是机理之一。
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 into
type 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 in
vitro, it’s induced by LPS(lipopolysaccharide,LPS). Under this model we’ll investigate the
alteration of alveolar type Ⅱ cells’ apoptosis、secretory function. At the same time we use a
traditional 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 been
improved, the cells puritied by adhered to IgG coated dishes and identified with a tannic acid
stain and AKP stain. The cellular ultra-structure in alveolar type Ⅱ cells were observed by
these two method.
In the second part, We add different doses of LPS from 10ug/ml to 100ug/ml in culture
medium to stimulate alveolar type Ⅱ cells for 2 hours. the concentrations of TNF- and IL-8 in
the culture media were determined based on ELISA . Fluorescence- quantitative-PCR technique
was performed to quantitate the levels of SP-A、SP-B and AQP-1mRNA in alveolar type Ⅱ
cells.
Results:
1. Isolating, puritying and identifying alveolar type Ⅱ cells of rats: We get the 109 cells
before purification and 107 cells after it. The trypan blue stain shows that the cells’ activity over
the 96 percent. Identified with a tannic acid stain and AKP stain, before purification the
puritying 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 of
LPS from 10ug/ml to 100ug/ml for 2 hours in culture medium, LPS could result in the necrosis
and 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 of
LPS from 10ug/ml to 100ug/ml for 2 hours in culture medium , LPS could stimulate secretion of
TNF-α of alveolar type Ⅱ cells significantly increased as compared with control group. After
use the medicine the increase will be inhibited obviously. The secretion of IL-8 have not a
significantly increase.
4. The expression of SP-A、B and AQP-1 in LPS injured type Ⅱcells was significantly
decreased as compared with control group,P<0.001。After use the traditional Chinese
medicine 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 the
concentration of digestive enzyme and digestion time could rise the cellular vitality. Adhered to
IgG coated dishes after isolating provided more high cell purity than before. It could be found
the 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 the
secretion of TNF-αof alveolar type Ⅱ cells significantly increased, and the expression of
SP-A、B andAQP-1 was significantly decreased.
4. After use the traditional medicine these injury are all inhibited. It is implicated that this
medicine might directly inhibit LPS induced injury of alveolar type Ⅱ in vitro.
引文
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综述二
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