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Repair effect of Qinbai Qingfei concentrated pellets to AEC-Ⅱ of rats infected by Mycoplasmal pneumonia

芩百清肺浓缩丸对肺炎支原体感染大鼠肺泡Ⅱ型上皮细胞修复作用的研究



全 文 :2016
y

z Ó
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Vol41,No.8 April,2016
·
k;
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fghi“”j¶ikl2m‰no·ip
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øù

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GV

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*H’ /01
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wx
] 
@•¶â_Ë(Ø-
β(TGFβ)»+9̹Q©¾0A(SPA)´ ,ùžÄ+X_­µ@,¹ºfghi“”j¶
ikl2m
(MP)
‰no·ipⅡqrs56Ätu¯°f±。 60G Wistaro·,\Ñx„,m;80~100g,1fµé6
´


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、¨
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PCR
#©!i´7$
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Õà

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ÖÄi´7)5¨*ºÜ:B[!Ätu¯°


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] 
ikl2m

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(81374045)

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] 
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Š‹ŒEhi¡
,Tel:(0451)55665478,Email:342201601@qqcom

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] 
øù

V

àŠŸh¨
,Tel:(0451)55665478,Email:yaoyao198003@aliyuncom
RepairefectofQinbaiQingfeiconcentratedpeletstoAECⅡ ofrats
infectedbyMycoplasmalpneumonia
YAOLin,ZHANGJunwei,WANGBo,SUIMeijiao,WANGWeiming
(HeilongjiangAcademyofChineseMedicalSciences,Harbin150036,China)
[Abstract] TodiscusstherepairmechanismofQinbaiQingfeiconcentratedpeletstoAECⅡ ofratsinfectedbymycoplasma
throughtheobservationofthechangesanddistributionofTGFβandSPAinlungs,totaly60Wistarratsthatweighing80100gwere
colected,withmaleandfemaleinhalfTheratsweredividedintosixgroupsrandomly,with10ratseachgroup,namelyblankgroup,
modelgroup,positivegroupandQinbaiQingfeiconcentratedpeletshigh,middleandlowdosegroupsRatswereinfectedthroughna
salintubationdripofMPAfter10daysofadministration,serumandbronchoalveolarlavagefluid(BALF)werecolectedtodetectthe
concentrationofsurfaceactivityrelatedproteinA(SPA)byELISA,leftpulmonarytissuesofratswerecolectedtoobservetheexpres
sionanddistributionoftransforminggrowthfactorbeta(TGFβ)andSPAbyimmunohistochemistry,andrightpulmonarytissueswere
takentodetectTGFβandSPAmRNAexpressionlevelbyrealtimequantitativePCR(RTPCR)QinbaiQingfeiconcentratedpelets
canreducetheexpressionofTGFβandincreasetheexpressionofSPAinthelungtissuesofratsinfectedbymycoplasmaSpecifical
ly,TGFβwasmainlydistributedamongthelunginterstitium,whileSPAsweremainlydistributedinAECIandpartsofalveolarmac
rophageThelevelofSPAwasreducedinserumandincreasedinBALFinratsinQinbaiQingfeiconcentratedpeletsgroupsItwas
provedthatQinbaiQingfeiconcentratedpeletscanrestorethenormalmorphologyandfunctionofthelungbyreducingthecontentof
TGFβtoinhibitepithelialmesenchymaltransition(EMT)ofalveolartypeIepithelialcelsandincreasingtheexpressionofSPA
·3941·
2016
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Vol41,No.8 April,2016
QinbaiQingfeiconcentratedpeletshavetherepairingabilitytocapilaryvesseldamagecausedbyMPinlungtissuesofrats
[Keywords] MycoplasmaPneumoniae;QinbaiQingfeiconcentratedpelets;AECⅡ;TGFβ;SPA
doi:10.4268/cjcmm20160821
  
ikl2m
(Mycoplasmapneumonia,MP)
æ=
ÖS“¬‰n©lÄÁ2m

øùÄÅr¶Þik
l2mik
(Mycoplasmapneumoniaepneumonia,
MPP)
ĺRª«ê°oM8ýG²ËF

fæãG
²ËFҏ4êo

y¯°î

¦#Ël2mÄUO
¶ÂÃÄfm¡Ê[!ĺÜ

S2,˜½æyr
¯Äµ¶
[1]。
Ø_

~m[,nÕç#Ël2må
Õ琑Vºi´7tuÄ,Éø›é;«

fghi“”j

çÀú

fg
”)
挡p
e$A,&®Èª…m

’%GKÎÄÓ[Y°
ÞÄŅ†ºR:4l2mikÄ$,ß,


f

g1



Ïñ

g+

áL

ï$,´Ê

½.
…†»ÄÅ89D®B–ÞIJ
MP,
ú·­t
uVºi´7Öʬ
[25]。
ipⅡqrs56(typeⅡ alveolarepithelial
cel,AECⅡ)æiprsċ56,æHÊ»µ%
ip+9̹Éô­®Q©¾0Ī«56
[6]。
MP
‰nMªž

=ÖĪ«Áè+×éJô¹i
k

{%
AECⅡVº,ÁáQ;Ý4um¯Õ=
ÖrsJôâ_
(epithelialtomesenchymaltransi
tion,EMT),
Trs56ùrsǹ

â_Ë(Ø
-
β(transforminggrowthfactorβ,TGFβ)æ[,U
Ø,56µ%Ä:BØ;ËɬêÄؚG56
Ø-

…†m×
,TGFβ¦ AECⅡmË EMT•–$
Ö2½©;«Ä˔¯°
[78]。AECⅡ ºÜ
ž
[910],
Õz{rs@¶¹c‘

›±i+9̹É
ôQ©¾0
A(surfactantassociatedproteinA,SPA)
ÄHÊ»µ%
,SPA
xõě±üi´7ùé
Ó#ô{%®ºÜ‘[¸‘;

u…†@••–´,ùž

ikl2m‰no
·i´7$
TGFβ» SPA+Xā_,)fg

AECⅡÄtu¯°,qí鮐‘i´7tu、
MuiÊöį°f±Ÿ/èÒ×ë

1 
XL
fghi“”j

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E

Î<
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u[éH!RčïýA“”‚
j

’¨/OH
2010


$%,.

j„ÀÄx
͝!

ikl2m%p‚`
(ATCCFH15531,
Û%
­,K$S
);PPLO
?É×
(Becton,Dickinson
andCompany,USA);TRIzol


Û%
Invitrogen,
Î
<
66211);TranscriptorfirststrandcDNASynthesis
Kit(Roche
DE

Î<
1485422);FastStartUniversal
SYBRGreenMaster(ROX)(Roche
DE

Î<
10277200);SPA,TGFβ,βactin=É(.BNOP
ËÉ()BCDE
);TGFβØI[²m(ABCAM
DE
);SPA
ØI[²m
(SANTA
DE
);SPA
¨©
B„¼

ëT¥ð“šB.#
,SEA890Ra96T,
L140724594,Uscn);
“”q
DAB
B„¼

$í$
Þ
);
ظ#¥ð´_B„¼

$í$Þ
)。
Wistar

,60
G

\Ñx„

m;
80~100g,
(iJà½.&É()BCDEŸ/
,SPF
/


D<
SCXK(

)20110004。
SorvaⅡ ST16±‰ªSf(ThermoDE);KD
TS3A
È&´7‰‚f

op$B&³ÚÃÿìB
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);KDBM
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op$B&³Ú
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);RM2235
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’%]
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);BX41
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(OLYMPUS
DE
);DP72
´
7ËÉv<“ŽµîÙ=
(OLYMPUS
DE
);Infi
niteM200Pro
ëÚ
(Tecan
DE
);9600

PCR
¨©Ú

¡¢O{
)。
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#
21 
&ɵ´

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 60
G
Wistar
o·1
fµé
6´ ,

10
G

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µeé$0¶#
´

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$

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´

o·ê¹ÒÉ



°
1%
÷m•ðŨ
©gu°ê

­$0¶#´£

®x´o·Lç8
åå i
1×106CCU·mL-1MP
­P
50μL,Ü°
®Èn“Ã!&¯-­P“iì“i1

$0¶
#´ iÖõ
MP
?ÉP

‰nžÓ

!~ÕÖ
´,

|{

Š

$0¶#´»Îq¶#´ÕÖ֝
ËËè܂

p¹¶#´ÕÖ}JÂF

ZŠ´,
õé
45mg·kg-1·d-1,
Ó
2~5

225mg·
kg-1·d-1),
ˆ#´,
5d;
fgO
(32g·kg-1·
d-1)、
$
(16g·kg-1·d-1)、
±
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„õ´ˆ#´,
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à 


fghi“”j¶ikl2m‰no·ipⅡqrs56tu¯°Ä…†
¨ª&¢ ¨

µª¨h
-80℃
&B

°Þ¨.¨
h$
SPA


”ŽipÕwP
-80℃
&Bì°

°Þ¨.ipÕwP
(BALF)
$
SPA


堘
i´7
4%
æøŸ!

°Þ¥ð´_¨©

堙i
´7$é

°ÞŸ 
RNA。
22 
o·i´7
TGFβ,SPA¾0¨©  æø
Ÿ!Äi´7

°¯ŒŠr‰‚

Øæå$¶

Š

ƒizt8‘nýu

tՀfrՀ

-sw3~Ät€RšÞh†ö¾
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,68℃
å‰Z8R€
2h,
“°Ø¸#¥ð´_
¨©

°ø4´89´7nRáÎ

23 
o·i´7
TGFβ,SPAmRNA¨© ç½
O_¤!õ
PCR
#¨©
TGFβ»SPAmRNAÄ+
X

Ÿ 
RNA,
°ëÚ¶
RNA
7r­“r‘n
©!

¥¼
RNA
“rËÌu
RNA
ÄmË

üx&
[—âãmÙVxÄ
RNA
õQU

-
RNA
—âã
é
cDNA,
ç—âãHÊÄ
cDNA
é΀

‘nèH
ë_4ê

=Éó¯
[1113]:TGFβr/5′GGCGGT
GCTCGCTTTGTA3′,
À/
5′GCCACTCAGGCGTAT
CAG3′;SPA
r/
5′TAAGTGCTGCCCTCTGACCT
3′,
À/
5′AGGAGCCATACATGCCAAAC3′;βactin
r/
5′CGTTGACATCCGTAAAGAC3′,
À/
5′TG
GAAGGTGGACAGTGAG3′。
24 
o·¨h­
BALF
$
SPA
Ä©!
 
“°
ELISA
#¨©

¥¼Ñ½.
,¨

BALF
&[µe
‹Œ
100
’
、1000
’ž©!

Y¯–óˆ#
ELISA
B„¼«v‘n

25 
=Dµî
 
“°
SPSS180
†p‘n=D
µî

=¼°珋x±s+H,2´ J‘=•–“°þc&
u
t¨
.

ç
P<005
éÚÛB=Dd<

3 
ä
31 
o·i´7
TGFβ,SPA¾0+XÄ¥ð´
_¨©
 
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ƒo
200
’

|´|!€-
€

YÙg

µe©|Y!R‰ùÄ
MOD,
vȖ
 Í‘^

°
IPP
†pµî

;Rvó

+Xvî

@•¶
TGFβ¾0+X¨©Õà,$0¶#´
o·i´7$¦iJô­ip6!9ÕlkõÄ
TGFβ¾0+X,¦ip¦8Õl(ÄñR²³,S
2R–¦

Ô$0´•–

Îq´o·iJô­ip
6!9Ä
TGFβ¾0+Xø,ÕloõHñR²
³

Áx8¨*

lÃ*!9µåÄ1µkÀ56

ipJƒ$ÄJô56‘BóñR²³

6¦8H
ñR²³2R–ó

µ@+Ž

YeÕlé<¾µ
@

:Bøù¹ÚÛ
(P<001);
ÔÎq´•–

f
gx„õ´
TGFβ¾0+XøÀ›(P<005),
SO„õ´À›ø

+
1,
q
1)。
+
1 
fg¶
MPP
o·i´7$
TGFβ,SPA¾0+XÄ
ÂÃ
(珋x±s,n=10)
Table1 TheefectofQinbaionTGFβandSPAexpression
levelinlungtissueofMPPrats(珋x±s,n=10)
µ´
„õ
/g·kg-1·d-1
TGFβ
/βactin
SPA
/βactin
$0
- 0173±0063 0384±0063
Îq
- 0430±01101) 0172±00151)
}JÂF ZŠ
0045 0292±00223) 0281±00593)
Ó
2~5
!
00225
fgO
32 0236±00133) 0234±00163)
fg$
16 0304±00262) 0201±00202)
fg±
08 0312±00282) 0171±0008
  
g

Ô$0´•–
1)P<001;
ÔÎq¶#´•–
2)P<005,
3)P<001(
+

U
)。
q
1 
x´o·i´7
TGFβ¾0+X(×20)
Fig1 TheproteinexpressionlevelofTGFβinlungtissueof
rats(×20)
  
@•¶
SPA
¾0+X¨©Õà

$0¶#´
Õl
SPA
+X§¯

+Žíó

µ@Þ
AECⅡ­1
µip’!568

½Hñ“H!R²³

ip­5
lÃ*©8Õl[8óHRÉô

Ô$0¶#´•
–

Îq¶#´
SPA
+Xô–µÅ

2R–¦

½
H!R²³

:Bøù¹ÚÛ
(P<001);
ÔÎq´
•–

fgO

$„õ´
SPA
+XŸO
(P<005),
Ձø89T
AECⅡ8HñR²³=õ»;Rc
Ø

ó

+
1,
q
2)。
·5941·
2016
y

z Ó
41
âÓ

}
Vol41,No.8 April,2016
q
2 
x´o·i´7
SPA
¾0+X
(×20)
Fig2 TheproteinexpressionlevelofSPAinlungtissueof
rats(×20)
32 
o·i´7
TGFβ,SPAmRNA+X ÔÎ
q´•–

fgx„õ´
TGFβ×Ø+XøÀ›
(P<005),
SO„õ´À›ø

í
SPA
×Ø+
Xøc‘
(P<005),
U&O„õ´+Xj‘
ø

+
2)。
+
2 
fg¶
MPP
o·i´7$
TGFβ,SPAmRNA+XÄ
ÂÃ
(珋x±s,n=10)
Table2 TheefectofQinbaionTGFβandSPAmRNAex
pressionlevelinlungtissueofMPPrats(珋x±s,n=10)
µ´
„õ
/g·kg-1·d-1
TGFβ
/βactin
SPA
/βactin
$0
- 0978±0023 0980±0029
Îq
- 4753±06411) 0263±00051)
}JÂF ZŠ
0045 2503±04083) 0560±00772)
Ó
2~5
!
00225
fgO
32 2493±04313) 0748±00473)
fg$
16 2637±04593) 0490±00512)
fg±
08 3708±08052) 0327±0043
33 
o·¨h­
BALF
$
SPA
Äxõ©!
 
Ô$
0¶#´•–

Îq´¨h$
SPA
xõørË
(P<001),
í
BALF
$
SPA
xõÀË
(P<005);
ÔÎq´Q•

p¹¶#´¨h$
SPA
xõ_
Òøù
,BALF
$
SPA
xõˆBrË

f‘f=D
d<

fgO

$

±x´¨h$
SPA
xõÀË
(P<005),BALF
$
SPA
xõc‘
(P<005)

+
3)。
4 
ºÒ
TGFβé6¦q+XÄ56Ø-,®p¹+X
  +3 fg¶ MPPo·¨h­ BALF$ SPAxõÄÂÃ
(珋x±s,n=10)
Table3 TheefectofQinbaionconcentrationofSPAinserum
andBALFofMPPrats(珋x±s,n=10)
µ´
„õ
/g·kg-1·d-1
¨h
/μg·L-1
BALF
/μg·L-1
$0
- 9635±0929 58058±6688
Îq
- 12446±07682) 45829±58281)
}JÂF ZŠ
0045 12515±1234 50156±6374
Ó
2~5
!
00225
fgO
32 8819±160394) 63681±124973)
fg$
16 9427±29343) 59571±60153)
fg±
08 10390±2158 57542±59744)
  
g

Ô$0´•–
1)P<005,2)P<001;
ÔÎq¶#´•–
3)P<005,4)P<001。
ZɽHñR²³¾µ@Þ6¦$

x´o·i´
7$
TGFβ+Xª«µ@ÞiJô,AlÞip
6

p¹+X²³ª«øÞ6ô8

;Rvó+Xv
î

@•Îq´Ô$0¶#´¶•ÕçSå
,MP
‰no·ž

UÞ£¿Ä{%
TGFβĵ%õ


ürs56îJô56Äâ

íx´,´
TGFβľ0+X›±,e,Éö÷@•›±
TGFβľ0+X™ÝkÀ4ê,y±rs56î
Jô56Ä⁕–

SPA
ª«µ@Þ
AECⅡ­1µip’!56
8

¥ð´_nRž½H!R²³

$0¶#´
SPA
µ@+Ž

S2R–ó

íÔ$0¶#´•–

Îq¶#´
SPA
+Xô–µÅ

2R–¦


MP
‰no·ž
,SPA
ľ0+X›±

ŸH
AEC
ⅡVº,i´7ùéÓ#,®™©ËèÊöVT
ÂÃ

ÔÎq¶#´•–

x´,´ö÷ŸO
SPA
ľ0+Xõ

e,ɶVºÄ
AECⅡ:Btu
į°

‘®µ%»HÊ
SPA,
Muię©Ë
èÊö

ü®‘i¹ÄË辿

MPP
o·i´7$
TGFβ­ SPAmRNAĨ
©ä+

o·‰n
MP
ži´7$
TGFβmR
NA
øùrË

í
SPAmRNA
+XÀ›

ŸH
MP
‰
n¦=ÖJô¹ikĕ–$Õc‘i´7$
TGFβĵ%,ŸO TGFβmRNA+X,‘ AEC
ⅡÄrsJôâ_;AECⅡmËrsJôâ_ž,
SPA
µ%™k
,SPAmRNA
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