| BackgroundUnder a variety of chronic liver injury, normal liver will cause persistent or recurrent liver parenchyma inflammation and necrosis, making the fibrous connective tissue proliferated, while its degradation of the relative lack of. A large number of extracellular cell matrix (ECM) deposit of the formatting liver fibrosis and further progress lead to cirrhosis. If giving effective treatment, the liver fibrosis can be reversed. HSC is activated by the resting state is the key link and cytological basis of liver fibrosis, activated HSC can synthesize and secrete the ECM, leading to ECM deposition in the liver of excess.TGFβ1plays an important role in liver fibrosis which is considered to be strongest fibrosing factor. It can promote HSC to transform fibroblast-like muscle cells, increase extracellular matrix production, reduce the matrix degradation, inhibit liver regeneration and induction of apoptosis.IGFBPrP1is a soluble secreted protein. Professor Lixin Liu discovered that IGFBPrP1was highly expressed in liver fibrosis and cirrhosis in patients with liver tissue, which was positively correlated with high expression of TGFβ1and Collagen I; After exogenous recombinant TGFβ1to stimulate HSC in vitro, it can increase the expression lelves of protein of IGFBPrPl; Anti IGFBPrPl antibody can inhibit the expression of TGFβ1of liver tissue in TAA-induced hepatic fibrosis. Studies have shown that0.05-0.1μg/L exogenous recombinant TGFβ1to stimulate the rabbit corneal endothelial cells, can promote cell proliferation. Endogenous TGFβ1gene can inhibite cell proliferation of rabbit corneal endothelial cells. Suggesting the different sources of TGFβ1on the same cell have different effects. To identify if the endogenous TGFβ1can influence IGFBPrPl synthesis with hepatic stellate cells in vitro, the subject is designed. O b j ec t i v eTo ide n ti fy if th e e nd o g e n o u s tr a nsfo rm in g g r ow t h f a c t or β1(T G Fβ1) c a n in flu e n c ein s u lin-lik e g ro w th fa c t o r bin d ing p ro te in re lat ed pr o t e i n1(I G F B P r P1) s yn t he s is w it h he p a t ics t e lla t e c e lls in vit ro.M et h o d s1. D e t e c t io n o f th e tr an s fe r c ti o n e ffi c ie nc yN e g a t iv e pl a s mid p EG FP-N1w it h flu o rescen ce w a s t r a ns f e c t e d in t o L X-2, a ft e r48h, w eo bs e r v e d th e tr a ns fe rc ti o n e ffic ie n c y us ing flu o rescen c e m i c r o s c o p y.2. Ov e re xp r e s s io n o f e nd o g e no us TGF β1L X-2C e lls lin e w a s d iv id e d in t o th re e g ro up s: bl a nk c o nt r ol g r o up: a d de d e qu a l a mo u n to f me d iu m w it ho ut se ru m a nd a nt ibi o t ics; neg at i ve p l a s mid g r o u p:t r a ns fe c t e d ne g a t iv ep la s mid; T G Fβ1ov e r–e x p re ssio n gr o up:t r a nsf ec t ed T G F β1o ve r–e x pr e s s io n pl a s mid.A ft e rd if f e r e n t ti me s (24h,48h,72h), Re a l-t ime PC R was us e d t o d e t e r min e th e e x pr e s s io n le ve ls o fmR N A o f T G Fβ1a n d C o lla ge n I. A ft er72h, Wes t e r n blo t w a s u s e d to d e t e rmine th ee xp r e s s io n le v e ls of p ro te in o f TG Fβ1a nd Co llag en I.3. I nf lu e nc e o f e nd o g e no us T GF β1on IGF BP rP1L X-2C e lls lin e w a s d e a lt w it h a s fo rm er. A ft er d i f f e r e nt ti me s (24h,48h,72h), R e a l-t imePC R w a s u s e d to d e t e rmin e th e e xpr e ss io n lev els of m R N A o f I G FB Pr P1. Af t e r72h, Wes t e rnblo t w a s us e d to d e te rm in e th e e xp re ss io n lev e ls of p r o t e i n o f I G F B Pr P1.Re s u l t s1. Tra ns f e r c ti o n e ffi c ie nc y48h la t e r,u nde r th e flu o re sc e n c e micr o sco py, th e g r e e n f lu o r e s c e nc e w it h th e c e lls c o u ldbe s e e n a ft e r tr a ns fe c t io n, su g ge st i ng th e tr a nsf ect io n w a s s u c c e s s f u l a n d th e tr a ns fe c t io ne ff ic ie nc y w a s40%.2. E x pr e s s io n le v e ls o f mR N A a nd pr ot e in o f TGF β1a n d C o lla ge n IT he re s u lt s o f R e a l-t ime PC R a naly si s:24h a f t e r t r a ns f e c t io n, th e e xp re s s io n le ve ls o fT G Fβ1ov e r-e xp re s s io n gr o up (32.22±0.39) o f TG F β1m R N A w e r e s ig ni f ic a n t ly hig h e r th a nth o s e in bla n k c on t ro l g ro up (1)a n d neg at iv e p la s m i d g r o u p(7.98±0.12)(P＜0.01). Thed if f e r e n c e o f C o lla g e n I mR N A e x pr e ssio n bet ween T G F β1o ve r-e xp r e s s io n g ro u p (1.92±0.04)a n d ne g a t ive p la s mid g ro u p(1.99±0.07) w as no t sig n i f i c a n t (P＞0.05).48h a f t e r tr a ns fe c t io n,th e e xp r e s s io n le v e ls o f TG Fβ1ov e r-e xp re ssi o n g r o up (63.12±0.44) of T G Fβ1mR N A w e res ig n if ic a n t ly hig h e r th a n th o se in blan k co nt ro l g r ou p (1) a n d ne g a t iv e p la s mid g ro u p (1.01±0.01), the expression levels of TGFβ1over-expression group(2.00±0.06) of Collagen I mRNA were significantly higher than those in blank control group(1) and negative plasmid group(1.02±0.01)(P<0.01).72h after transfection, the expression levels of TGFβ1over-expression group(38.46±0.19) of TGFβ1mRNA were significantly higher than those in blank control group(1) and negative plasmid group(1.05±0.01), the expression levels of TGFβ1over-expression group(8.63±0.15) of Collagen I mRNA were significantly higher than those in blank control group(1) and negative plasmid group(1.03±0.02)(P<0.01). The results of Western blot analysis:72h after transfection, the expression levels of TGFβ1over-expression group(0.27±0.04) of TGFβ1protein were significantly higher than those in blank control group(0.20±0.01) and negative plasmid group(0.22±0.01), the expression levels of TGFβ1over-expression group(1.47±0.02) of Collagen I protein were significantly higher than those in blank control group(0.76±0.03) and negative plasmid group(0.77±0.02).3. Expression levels of mRNA and protein of IGFBPrPlThe results of Real-time PCR analysis:24h after transfection, the expression levels of TGFβ1over-expression group(4.33±0.38) of IGFBPrPl mRNA were significantly higher than those in blank control group(1) and negative plasmid group(2.86±0.09);48h after transfection, the expression levels of TGFβ1over-expression group(1.41±0.11) of IGFBPrP1mRNA were significantly higher than those in blank control group(l) and negative plasmid group(1.07±0.16);72h after transfection, the expression levels of TGFβ1over-expression group(2.56±0.30) of IGFBPrPl mRNA were significantly higher than those in blank control group(1) and negative plasmid group(1.00±10.10)(P<0.01). The results of Western blot analysis:72h after transfection, the expression levels of TGFβ1over-expression group(0.53±0.01) of IGFBPrPl protein were significantly higher than those in blank control group(0.25±0.02) and negative plasmid group(0.27±0.03)(P<0.05).ConclusionEndogenous TGFβ1can increase the synthesis of mRNA and protein of TGFβ1and Collagen I with LX-2and also can promote the expression of mRNA and protein of IGFBPrP1. Speculating that IGFBPrP1have closely related with TGFβ1to regulate the process of liver fibrosis. |
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