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标题: Angiotensin II Ca2 signaling in rat afferent arterioles: stimulation of cyclic [打印本页]

作者: 轻羽    时间: 2009-4-21 13:03     标题: Angiotensin II Ca2 signaling in rat afferent arterioles: stimulation of cyclic

Department of Cell and Molecular Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina5 N' l4 m# J4 a7 @3 w( I' p% V

: i- ]  i/ X7 B- R7 u  s! ^ABSTRACT
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+ b- s" y9 k8 vANG II induces a rise in cytosolic Ca2  ([Ca2 ]i) in vascular smooth muscle (VSM) cells via inositol trisphosphate receptor (IP3R) activation and release of Ca2  from the sarcoplasmic reticulum (SR). The Ca2  signal is augmented by calcium-induced calcium release (CICR) and by cyclic adeninediphosphate ribose (cADPR), which sensitizes the ryanodine-sensitive receptor (RyR) to Ca2  to further amplify CICR. cADPR is synthesized from -nicotinamide adenine dinucleotide (NAD ) by a membrane-bound bifunctional enzyme, ADPR cyclase. To investigate the possibility that ANG II activates the ADPR cyclase of afferent arterioles, we used inhibitors of the IP3R, RyR, and ADPR cyclase. Afferent arterioles were isolated from rat kidney with the magnetized microsphere and sieving technique and loaded with fura-2 to measure [Ca2 ]i. In Ca2 -containing buffer, ANG II increased [Ca2 ]i by 125 ± 10 nM. In the presence of the IP3R antagonists TMB-8 and 2-APB, the peak responses to ANG II were reduced by 74 and 81%, respectively. The specific antagonist of cADPR 8-Br ADPR and a high concentration of ryanodine (100 μM) inhibited the ANG II-induced increases in [Ca2 ]i by 75 and 69%, respectively. Nicotinamide and Zn2  are known inhibitors of the VSM ADPR cyclase. Nicotinamide diminished the [Ca2 ]i response to ANG II by 66%. In calcium-free buffer, Zn2  reduced the ANG II response by 68%. Simultaneous blockade of the IP3 and cADPR pathways diminished the [Ca2 ]i response to ANG II by 83%. We conclude that ANG II initiates Ca2  mobilization from the SR in afferent arterioles via the classic IP3R pathway and that ANG II may lead to activation of the ADPR cyclase to form cADPR, which, via its action on the RyR, substantially augments the Ca2  response.
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calcium-induced calcium release; ryanodine; vascular smooth muscle; nicotinamide0 F* f2 O* g& l/ }( S

/ ?$ Z. B- N8 }9 ?' tIT IS BECOMING generally accepted that agonist stimulation of G protein-coupled receptors, formation of IP3, and activation of IP3 receptors (IP3R) cause release of Ca2  of very short duration from the endoplasmic and sarcoplasmic reticulum (ER/SR) (3, 26). The Ca2  signal, likely initiated by IP3, is then amplified and sustained by activation of a ryanodine receptor (RyR) by the increase in cytolsolic Ca2  ([Ca2 ]i). This process, known as calcium-induced calcium release (CICR), is enhanced by cyclic adeninediphosphate ribose (cADPR) and possibly by IP3 as well (22, 26, 53). cADPR, discovered by H. C. Lee in 1989 in sea urchin eggs (38), is synthesized from -nicotinamide adenine dinucleotide (NAD ) by a bifunctional membrane-bound enzyme, ADP ribosyl cyclase, in a wide variety of eukaryotic cells (24, 36). cADPR activates a RyR (40, 54) and, in conjunction with calmodulin, sensitizes a RyR to Ca2 , thereby augmenting CICR (14, 23, 34).
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' S  H4 Y9 S" G. P  mEvidence for the presence of an ADPR cyclase and for a role of cADPR in Ca2  signaling has been established in a large number of mammalian cell types, including pancreatic islet cells, cardiac myocytes, trachea, intestinal longitudinal muscle, lymphocytes, sympathetic neurons, salivary and lacrimal gland cells, and hepatocytes (24, 36). Only recently, however, has a role for cADPR been investigated in vascular smooth muscle (VSM). In membrane preparations of aorta (13, 53), renal microvessels (39), coronary arteries (31, 52, 55), and pulmonary artery (16), evidence for ADPR cyclase activity has been demonstrated. Measurement of changes in [Ca2 ]i in response to cADPR has been made in permeabilized renal VSM cells (39). To our knowledge, there have been no Ca2  studies examining the activation of the cADPR pathway in intact fresh afferent arterioles and no studies exploring the effect of ANG II on this particular pathway in VSM of any origin.5 v) E% m+ ?8 ~$ m

, {9 s8 g% U0 h. ]The ADPR cyclase of VSM has several unique properties that distinguish it from the CD38 ADPR cyclase of nonvascular cells. In contrast to the CD38 enzyme of sea urchin eggs, aplysia, and HL-60 cells, in which Zn2  enhances the activity of the enzyme, Zn2  inhibits the cyclase of rat aortic VSM cells (13). Nitric oxide (NO) inhibits the VSM enzyme, whereas it is stimulatory in macrophages, neurons, pancreatic cells, and sea urchin eggs (52). Recently, it has been shown that oxidative stress increases [Ca2 ]i in fresh bovine coronary VSM cells through a pathway that involves cADPR (55) and that NO inhibits ADPR cyclase in coronary artery VSM (52).4 \9 e# ?7 q/ L" y

1 W6 w" c2 I7 d* b) f9 V$ YOnly one study investigated the effect of ANG II on the activity of ADP-ribosyl cyclase (27). This laboratory found that in membrane preparations of neonatal but not older cardiac myocytes, ANG II increased cyclase activity in a dose-dependent fashion (27). The mechanism by which ANG II stimulated an increase in ADPR cyclase activity is unknown, but these investigators speculated that a G protein-coupled process is involved (27). Because of the crucial importance of afferent arterioles in regulating glomerular filtration and sodium balance, we investigated the effects of ANG II on Ca2  signaling in freshly isolated afferent arterioles using inhibitors of IP3R, RyR, and ADPR cyclase.
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METHODS$ v% W( f8 c, C: I

2 d# K, |) l: L# ?8 H' D6 ~All studies were performed in compliance with the guidelines and practices of the University of North Carolina at Chapel Hill Institutional Animal Care and Use Committee.4 P( N3 M* p- A( R6 r! Q
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Preparation of fresh afferent arterioles. We used the magnetized polystyrene microsphere sieving technique as previously described in our laboratory (20) to isolate afferent arterioles (+ u9 _3 Q3 j, G" B7 ^4 U3 x

( @# h; J* g8 v6 Y9 B% B$ J3 C* jVessels were stimulated with ANG II (3 x 10–7 M) (18). The concentrations of antagonists we chose were based on published results: 8-(N,N-diethylamino) octyl 3,4,5-trimethoxybenzoate (TMB-8) (44, 47), 2-aminoethoxydiphenyl borate (2-APB) (41, 42), ryanodine (7, 18), Zn2  (13), and nicotinamide (46). A dose-response analysis was performed for 8-Br cADPR (51). Arterioles were incubated with inhibitors for at least 1 min before initiation of an experimental measurement.
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  U4 ^0 p/ s9 ]8 f2 VMeasurement of [Ca2 ]i. We measured [Ca2 ]i as previously described (20). Afferent arterioles were identified by their morphology and measured diameter of 15–20 μm. Additionally, we required visualization of microspheres in the lumen to exclude the possibility that the vessel was an efferent arteriole. The microspheres (4.0–4.5 μm) do not pass beyond the glomerular capillaries. An arteriole was centered in a small window of the optical field that was free of glomeruli or tubular fragments.- K+ S: h9 X. e% w) T% @9 `- _3 I

( F/ a! B# C9 i( w7 gAlthough endothelial cells are present in these nonperfused afferent arterioles, we and others assumed that VSM cells provide the major source of fura-2 fluorescence owing to the greater bulk of VSM cells compared with endothelial cells (9). Studies in deendothelialized renal microvessels showed that ANG II-induced Ca2  increases were not different from those of control arterioles (43). Furthermore, all reagents were added to the bath and therefore there was no immediate intraluminal exposure of endothelial cells to agonists and antagonists. We showed previously that freshly isolated single preglomerular VSM cells and afferent arterioles respond similarly, both qualitatively and quantitatively, to endothelin (20).
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# J' J  z$ u4 J, WThe VSM cells were excited alternately with light of 340- and 380-nm wavelength from a dual-excitation wavelength Delta-Scan equipped with dual monochronometers and a chopper [Photon Technology International (PTI)] as previously described (17–19). After passing signals through a barrier filter (510 nm), fluorescence was detected by a photomultiplier tube. Signal intensity was acquired, stored, and processed by an IBM-compatible Pentium computer and Felix software (PTI). Background subtraction was performed in all studies. There was no interruption in the recording during the addition of reagents to the chamber. A video camera projected images of afferent arterioles onto a video monitor permitting visualization of contraction of vessel segments.6 I5 O3 c: K8 |7 ]& E7 n1 O' |

- W* s4 p# L4 w) s& H5 bReagents. We purchased ANG II, 2-APB, TMB-8, 8-Br cADPR, and nicotinamide from Sigma (St. Louis, MO), collagenase type IV from Worthington (Lakewood, NJ), fura-2AM from Molecular Probes (Eugene, OR), and magnetized microspheres from Spherotech (Libertyville, IL).
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Statistics. Data are presented as means ± SE. Each data set was derived from afferent arterioles originating from at least three separate experimental days, two rats (4 kidneys) per experiment. Individual arterioles were studied only once and then discarded. In experiments with inhibitors, values for ANG II peak responses were included only for the experimental days in which a particular inhibitor was employed. Paired data for arterioles before and after ANG II stimulation were tested with Student's paired t-test. Unpaired t-tests were employed for comparisons of responses between groups.
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2 G( H1 C" M. T6 O1 ~- d* o# f3 ?[Ca2 ]i response to ANG II. Afferent arterioles in Ca2 -containing PBS responded to ANG II with a sharp peak response in 1–3 s. The mean baseline [Ca2 ]i was 64 ± 4, the peak 189 ± 10, and the plateau 100 ± 7 nM (n = 48, P
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Effect of inhibitors of the IP3R. To assess the contribution of G protein-coupled ANG II receptor stimulation of phospholipase C (PLC), formation of IP3, and IP3R-mediated release of [Ca2 ]i from the SR, we employed two inhibitors of the IP3R, TMB-8 and 2-APB (42, 47). Because these studies were conducted in Ca2 -containing buffer, inhibition of IP3R-mediated Ca2  mobilization may also affect Ca2  entry mechanisms (e.g., voltage-gated Ca2  and store-operated Ca2  entry channels) as well. Approximately one-half to two-thirds of the initial peak Ca2  responses to ANG II in afferent arterioles have been shown to arise from mobilization mechanisms (5, current study, vide infra). Afferent arterioles were stimulated with ANG II in the absence (n = 11) or presence (n = 8) of TMB-8 (10–5 M; P  0.5 for both agents). These data confirm that ANG II-stimulated IP3 production and activation of the IP3R-mediated Ca2  release from the SR contribute substantially to the initial Ca2  response.
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Effect of inhibitors of the RyR. We postulated that blockade of RyRs should abolish CICR (4) and the ability of cADPR to enhance the Ca2  sensitivity of RyRs (24, 36). Ryanodine in high concentrations locks the RyR in a closed state (7, 48). Previously, we showed that a low concentration of ryanodine (2–3 μM) activates the RyR to mobilize Ca2  from the SR (18). In contrast, a high concentration of ryanodine (100 μM) had no effect on baseline [Ca2 ]i (21). In the current study, we found that ryanodine (100 μM) did not alter basal [Ca2 ]i in afferent arterioles but inhibited the subsequent ANG II response by 69%. As Fig. 3C shows, the average increase in [Ca2 ]i following stimulation with ANG II alone was 163 ± 27 nM (n = 12) and with ANG II following ryanodine was 50 ± 10 nM (n = 8, P ; H0 ^) w2 p% s  Z! `' e# w/ K: F
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We tested the ability of 8-Br cADPR, a cell-permeant antagonist of the RyR (36), to attenuate the Ca2  response to ANG II. In a dose-response analysis, 8-Br cADPR at concentrations of 50 and 100 μM was equally inhibitory (ANG II-induced increases in [Ca2 ]i were 38 ± 5 and 31 ± 5 nM, respectively, P  0.3). A representative tracing is shown in Fig. 3B. In this group of experiments, ANG II alone increased [Ca2 ]i by 152 ± 17 nM (n = 11), but in the presence of 8-Br cADPR, the net increase was only 36 ± 5 nM (76% inhibition, n = 16, P : s1 n. r" D  |
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Inhibitors of the ADPR cyclase. Thus a major question in trying to understand the relationship between agonist stimulation of a G protein-coupled receptor to initiate the sequence of IP3 generation, activation of the IP3R, release of Ca2  from the SR, and participation of the RyR to augment the Ca2  signal is what is the communication to the membrane ADPR cyclase to direct formation of cADPR We tested the effect of nicotinamide and Zn2 , well-studied inhibitors of the ADPR cyclase of VSM (13, 46), on Ca2  signaling in response to ANG II. Nicotinamide (3 mM) pretreatment of afferent arterioles did not change baseline values of [Ca2 ]i but reduced the [Ca2 ]i response to ANG II by 66% (Fig. 4A). In this group of experiments, ANG II alone increased [Ca2 ]i by 102 ± 18 nM (n = 7); in the presence of nicotinamide, the response was reduced to 35 ± 6 nM (n = 9, P
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As noted above, Zn2  inhibits the VSM ADPR cyclase, whereas it stimulates the cyclase of nonvascular cells (13). Because Zn2  may inhibit voltage-gated Ca2  entry channels (32) and because voltage-gated Ca2  entry is a major entry pathway in afferent arterioles (6, 10, 29), our studies assessing the action of Zn2  on the ADPR cyclase were done in Ca2 -free buffer. A typical tracing (Fig. 4B) shows that the response to ANG II is attenuated in Ca2 -free PBS and is markedly inhibited by Zn2  (3 mM). In nominally Ca2 -free PBS, ANG II increased [Ca2 ]i by 77 ± 30 nM (a value that is 62% of the response of afferent arterioles to ANG II in Ca2 -replete PBS). In the presence of Zn2 , the response to ANG II was decreased to 25 ± 10 nM (n = 7 for both, P 3 W4 X7 j% Q( P& Y( Y! v; S- h

- k9 z% x4 Y; ?& bSimultaneous inhibition of ADPR cyclase and of IP3R. To further document the relative contributions of activation of the IP3R and of formation of cAPPR, which sensitizes the RyR to Ca2 , we employed inhibitors of both of these pathways. In this group of experiments, ANG II increased [Ca2 ]i by 100 ± 13 nM (n = 16). In afferent arterioles pretreated with nicotinamide and 2-APB, the increase in [Ca2 ]i following addition of ANG II was 18 ± 4 nM and with nicotinamide and TMB-8 17 ± 3 nM (n = 9 and 8, respectively, P
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DISCUSSION+ B# f, n) t. d# y

$ ~+ y; u6 c1 {( FIn the present study, we show that G protein-coupled receptor activation by ANG II leads to Ca2  release from the SR via two different receptor/release channels, one sensitive to IP3 and the other to ryanodine. We present new information that ANG II signaling in fresh afferent arterioles causes activation of the VSM ADPR cyclase and formation of cADPR. The mechanism by which this occurs awaits elucidation. Only one study, performed in neonatal rat cardiac myocytes, suggests that ANG II (possibly with involvement of a G protein) activates an ADPR cyclase to form cADPR, which, in turn, stimulates the RyR to release Ca2  from the SR (27).
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We were the first to demonstrate that fresh preglomerular VSM cells have a functional RyR (18) and that depletion of SR Ca2  stores with ryanodine activates store-operated Ca2  entry in both Wistar-Kyoto and spontaneously hypertensive rats (18, 19). Subsequently, other laboratories presented evidence for a role for a RyR in renal VSM. Stimulation of cultured renal VSM cells with RGD peptides induces Ca2  waves that are completely blocked by closure of the RyR with ryanodine (20 μM) but not by the IP3R antagonist xestospongin (8). In the isolated hydronephrotic kidney model, ANG II-induced arteriolar oscillations in afferent arterioles are blocked by ryanodine but not by 2-APB, an IP3R inhibitor (50). In freshly dispersed VSM cells derived from third and fourth order renal vessels, the IP3R and RyR-sensitive Ca2  stores of the SR appear to communicate with each other, in contrast to the spatial organization of pulmonary arterial VSM cells in which the compartments are independent (30). Taken together, these studies confirm the important physiological role of RyRs in renal VSM.
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0 S# R3 E2 E7 @$ VIn the present study, we examined ANG II-stimulated participation of cADPR by inhibiting the ADPR cyclase and by assessing the contribution of cADPR to the stimulation of RyRs. When RyR is locked in a nonconductive state with a high concentration of ryanodine (100 μM) (7, 48), the [Ca2 ]i response of afferent arterioles to ANG II is markedly reduced. Pretreatment of arterioles with the cell-permeant antagonist of cADPR, 8-Br cADPR, likewise substantially diminishes the response to ANG II. Thus ANG II appears to be involved in stimulating the formation of cADPR. Taken together, our data are consistent with the conclusion that the effect of cADPR on the RyR and the participation of cADPR in sensitizing the RyR to Ca2  (CICR) are major components of the total [Ca2 ]i response to ANG II stimulation of afferent arteriolar VSM and suggest that Ca2  release from the SR through the IP3R accounts for less than one-third of the mobilization pathway.
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, J. e  C5 O) Q) f1 kTo examine the possibility that ANG II influences the activity of the ADPR cyclase, we used two inhibitors of this plasmalemmal membrane enzyme, nicotinamide (46) and Zn2  (13). Nicotinamide does not actually inhibit the cyclase but rather forces the reaction in the reverse direction to form NAD  rather than cADPR (33). Nicotinamide is not known to influence other components of ANG II signaling pathways. Because Zn2  may inhibit voltage-gated Ca2  entry (32), a major entry channel in afferent arterioles (6, 10, 29), the Zn2  experiments were performed in Ca2 -free buffer. Zn2  may also inhibit the membrane Ca2  ATPases (28). If this were a significant effect in renal VSM cells, one would expect an enhancement of the [Ca2 ]i response to ANG II rather than inhibition. Zn2  is also an inhibitor of proton currents generated via NOX in phagocytic cells (11). Whether Zn2  has an interaction with the novel isoforms of NOX of VSM (2) has not been studied. Superoxide dismutase, which converts superoxide to H2O2, is inhibited by Zn2  (15), an effect that would enhance rather than diminish a possible role for superoxide in ANG II signaling. The strong inhibitory effects of nicotinamide and Zn2  in our studies provide evidence that ANG II stimulation of VSM of afferent arterioles ultimately results in activation of the ADPR cyclase to form cADPR." G/ E3 Z) ^1 X! n6 K

8 ~* t4 V+ G; w+ r+ ~$ c" ?An enzyme capable of forming cADPR was first described in homogenates of sea urchin eggs (38) and subsequently has been shown to be present in a wide variety of cell types (24, 25, 35). In mammals, a single bifunctional protein, CD38, can act as a cyclase or hydrolase for cADPR (37, 45). The ADPR cyclase of VSM appears to have several unique characteristics that distinguish it from CD38 (13). Of particular interest is the fact that the VSM enzyme is inhibited, rather than stimulated, by Zn2  (13).
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8 C+ z% q. N8 GMammalian ADPR cyclases of specific cell types are stimulated by a number of different agonists: for example, estrogen in myometrium (1), glucose in pancreatic beta cells (49), retinoic acid and triiodothyronine in aortic VSM cells (12), reactive oxygen species in bovine coronary VSM (55), ANG II in neonatal cardiac myocytes (27), tumor necrosis factor- and interleukin-1 in glomerular mesangial cell (54), and acetylcholine and endothelin in airway smooth muscle (51). How this structurally diverse group of molecules mediates the same process, namely activation of the ADPR cyclase, has not yet been elucidated with certainty.
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2 V! V0 g; U2 ^2 l& w3 I7 @3 k! LIn summary, we show that ANG II stimulation of afferent arterioles increases [Ca2 ]i via several distinct pathways. The classic G protein-coupled receptor activation that results in IP3 generation and release of [Ca2 ]i from the SR likely provides an initial and transient burst of [Ca2 ]i, which can activate CICR from RyR. We present new information demonstrating that the ANG II response is blocked by the specific cADPR antagonist, 8-Br cADPR, and by high concentrations of ryanodine, which closes RyR. At present, we do not know the pathway(s) that link activation of the ADPR cyclase to ANG II stimulation of afferent arteriolar VSM, studies of which await further investigation.
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GRANTS
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/ c" d& q6 B. h4 ]- N  A0 nThis work was supported in part by a grant from the Thomas H. Maren Foundation and from National Heart, Lung, and Blood Institute Grant HL-02334., p0 l2 R2 q3 p. J  \' e( E

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1 o9 m, D8 Q( I  Q: c1 f. O6 g# i! ?The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.0 b$ h+ l) H: h: O/ q( j
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Galione A, Lee HC, and Busa WB. Ca2 -induced Ca2  release in sea urchin egg homogenates: modulation by cyclic ADP-ribose. Science 253: 1143–1146, 1991.$ G' J# s5 X8 g# z- P

+ U& f, i& H/ yGuse AH. Cyclic ADP-ribose: a novel Ca2 -mobilising second messenger. Cell Signal 11: 309–316, 1999.( q1 k) t  C6 U9 A

: H8 I( w$ \( v5 iGuse AH. Regulation of calcium signaling by the second messenger cyclic adenosine diphosphoribose (cADPR). Curr Mol Med 4: 239–248, 2004.
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) m7 Y/ W+ {" f$ Y2 wHigashida H, Zhang J, Hashii M, Shintaku M, Higashida C, and Takeda Y. Angiotensin II stimulates cyclic ADP-ribose formation in neonatal rat cardiac myocytes. Biochem J 352: 197–202, 2000.7 R* D- ^* h+ Q4 I

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作者: 红旗    时间: 2015-5-22 12:08

干细胞行业  
作者: 昕昕    时间: 2015-6-16 06:53

呵呵 高高实在是高~~~~~  
作者: txxxtyq    时间: 2015-7-22 09:26

楼上的稍等啦  
作者: 舒思    时间: 2015-9-11 13:00

这个贴好像之前没见过  
作者: 依旧随遇而安    时间: 2015-10-6 16:34

@,@..是什么意思呀?  
作者: biobio    时间: 2015-10-11 12:18

回复一下  
作者: 我心飞翔    时间: 2015-11-1 14:26

我的妈呀,爱死你了  
作者: s06806    时间: 2015-11-8 19:18

小心大家盯上你哦  
作者: immail    时间: 2015-11-15 13:49

每天早上起床都要看一遍“福布斯”富翁排行榜,如果上面没有我的名字,我就去上班……  
作者: dypnr    时间: 2015-11-25 15:11

端粒酶研究
作者: 命运的宠儿    时间: 2015-12-14 14:18

感謝樓主 干细胞之家真的不错  
作者: nauticus    时间: 2015-12-15 12:34

真的有么  
作者: immail    时间: 2015-12-15 19:01

知道了 不错~~~  
作者: 罗马星空    时间: 2015-12-20 10:35

朕要休息了..............  
作者: xuguofeng    时间: 2015-12-25 14:10

几头雾水…  
作者: 张佳    时间: 2016-1-26 16:05

拿分走人呵呵,楼下继续!
作者: yukun    时间: 2016-1-28 20:54

一个有信念者所开发出的力量,大于99个只有兴趣者。  
作者: 依旧随遇而安    时间: 2016-2-25 18:18

不错不错.,..我喜欢  
作者: dglove    时间: 2016-3-24 22:42

帮你项项吧  
作者: 锦锦乐道    时间: 2016-4-13 18:34

帮你项项吧  
作者: MIYAGI    时间: 2016-4-18 07:26

偶啥时才能熬出头啊.  
作者: 我学故我思    时间: 2016-5-1 16:18

好困啊  
作者: bluesuns    时间: 2016-5-11 11:54

对不起,我走错地方了,呵呵  
作者: 泡泡鱼    时间: 2016-5-23 19:41

彪悍的人生不需要解释。  
作者: dongmei    时间: 2016-5-27 19:40

干细胞从业人员  
作者: laoli1999    时间: 2016-5-28 15:36

厉害!强~~~~没的说了!  
作者: ines    时间: 2016-7-19 14:35

谢谢分享了!  
作者: 快乐小郎    时间: 2016-8-1 20:56

不管你信不信,反正我信  
作者: 狂奔的蜗牛    时间: 2016-8-4 11:54

呵呵 那就好好玩吧~~~~  
作者: leeking    时间: 2016-8-6 00:52

做对的事情比把事情做对重要。  
作者: 甘泉    时间: 2016-8-13 12:42

我在顶贴~!~  
作者: DAIMAND    时间: 2016-8-13 17:58

做一个,做好了,请看  
作者: dr_ji    时间: 2016-9-7 20:09

今天临床的资料更新很多呀
作者: leeking    时间: 2016-10-2 19:35

不知道说些什么  
作者: 科研人    时间: 2016-10-9 19:52

快毕业了 希望有个好工作 干细胞还是不错的方向
作者: 983abc    时间: 2016-11-3 21:43

看完了这么强的文章,我想说点什么,但是又不知道说什么好,想来想去只想  
作者: 一个平凡人    时间: 2016-11-6 18:25

小生对楼主之仰慕如滔滔江水连绵不绝,海枯石烂,天崩地裂,永不变心.  
作者: changfeng    时间: 2016-11-23 08:09

不错,看看。  
作者: vsill    时间: 2016-11-27 13:10

看或者不看,贴子就在这里,不急不忙  
作者: 蚂蚁    时间: 2016-12-3 14:27

我想要`~  
作者: haha3245    时间: 2017-1-12 17:27

知道了 不错~~~  
作者: highlight    时间: 2017-1-15 20:33

勤奋真能造就财富吗?  
作者: 修复者    时间: 2017-1-26 16:54

干细胞研究非常有前途
作者: biodj    时间: 2017-1-31 02:54

希望大家都有好运  
作者: dglove    时间: 2017-2-5 14:52

顶的就是你  
作者: 蝶澈    时间: 2017-2-10 11:18

感谢党和人民的关爱~~~  
作者: ikiss    时间: 2017-2-13 10:35

做一个,做好了,请看  
作者: 与你同行    时间: 2017-2-20 17:18

我的啦嘿嘿  
作者: 狂奔的蜗牛    时间: 2017-2-27 10:01

给我一个女人,我可以创造一个民族;给我一瓶酒,我可以带领他们征服全世界 。。。。。。。。。  
作者: 三星    时间: 2017-2-28 17:57

真是有你的!  
作者: 依旧随遇而安    时间: 2017-3-1 22:33

角膜缘上皮干细胞
作者: 榴榴莲    时间: 2017-4-2 07:22

站个位在说  
作者: 依旧随遇而安    时间: 2017-4-6 13:54

谢谢哦  
作者: 石头111    时间: 2017-4-17 00:53

你加油吧  
作者: 三星    时间: 2017-4-19 02:27

每天早上起床都要看一遍“福布斯”富翁排行榜,如果上面没有我的名字,我就去上班……  
作者: 生物小菜鸟    时间: 2017-4-26 12:01

不错不错,我喜欢看  
作者: netlover    时间: 2017-5-5 18:01

呵呵,等着就等着....  
作者: aakkaa    时间: 2017-5-12 06:45

楼主,支持!  
作者: dada    时间: 2017-5-20 09:28

又看了一次  
作者: ringsing    时间: 2017-5-31 10:10

只有一条路不能选择——那就是放弃的路;只有一条路不能拒绝——那就是成长的路。  
作者: biopxl    时间: 2017-6-8 05:31

不是吧  
作者: 123456zsz    时间: 2017-6-21 05:02

干细胞疾病模型
作者: xiao2014    时间: 2017-7-9 08:27

…没我说话的余地…飘走  
作者: ikiss    时间: 2017-7-29 07:10

小生对楼主之仰慕如滔滔江水连绵不绝,海枯石烂,天崩地裂,永不变心.  
作者: keanuc    时间: 2017-8-1 15:43

挺好啊  
作者: renee    时间: 2017-8-8 09:18

哈哈,这么多的人都回了,我敢不回吗?赶快回一个,很好的,我喜欢  
作者: heart10    时间: 2017-8-29 05:46

站个位在说  
作者: ladybird    时间: 2017-9-7 06:57

好人一个  
作者: 陈晴    时间: 2017-9-26 04:05

今天的干细胞研究资料更新很多呀
作者: 大小年    时间: 2017-10-3 02:27

谢谢干细胞之家提供资料
作者: 加菲猫    时间: 2017-10-7 23:35

对不起,我走错地方了,呵呵  
作者: ladybird    时间: 2017-10-8 06:34

角膜缘上皮干细胞
作者: syt7000    时间: 2017-10-16 12:09

呵呵,支持一下哈  
作者: 再来一天    时间: 2017-10-24 21:27

今天临床的资料更新很多呀
作者: frogsays    时间: 2017-10-30 01:35

谁能送我几分啊  
作者: 老农爱科学    时间: 2017-11-8 02:01

照你这么说真的有道理哦 呵呵 不进沙子馁~~~  
作者: 生物小菜鸟    时间: 2017-11-15 07:41

貌似我真的很笨????哎  
作者: doc2005    时间: 2017-11-27 04:09

长时间没来看了 ~~  
作者: mk990    时间: 2017-12-9 10:18

问渠哪得清如许,为有源头活水来。  
作者: MIYAGI    时间: 2017-12-21 10:54

老大,我好崇拜你哟  
作者: 3344555    时间: 2017-12-30 13:26

干细胞治疗  
作者: syt7000    时间: 2018-1-28 04:57

生殖干细胞
作者: nauticus    时间: 2018-2-21 03:18

楼主good  
作者: SCISCI    时间: 2018-3-6 14:01

我卷了~~~~~~~  
作者: 锦锦乐道    时间: 2018-3-7 05:28

挤在北京,给首都添麻烦了……  
作者: 甘泉    时间: 2018-3-10 16:00

哦...............  
作者: 依旧随遇而安    时间: 2018-4-8 19:37

嘿嘿......哈哈......呵呵.....哟~呼  
作者: cjms    时间: 2018-4-22 15:42

肿瘤干细胞
作者: 生物小菜鸟    时间: 2018-4-23 18:15

加油啊!!!!顶哦!!!!!  
作者: youngcell    时间: 2018-5-4 11:43

很好!很强大!  
作者: 石头111    时间: 2018-5-6 21:10

顶你一下.  
作者: pspvp    时间: 2018-5-11 18:00

文笔流畅,修辞得体,深得魏晋诸朝遗风,更将唐风宋骨发扬得入木三分,能在有生之年看见楼主的这个帖子。实在是我三生之幸啊。  
作者: heart10    时间: 2018-5-12 02:24

好困啊  
作者: syt7000    时间: 2018-5-13 05:32

干细胞之家 我永远支持
作者: 安安    时间: 2018-5-14 09:27

dddddddddddddd  
作者: 温暖暖    时间: 2018-6-13 07:01

不错的东西  持续关注  
作者: 小倔驴    时间: 2018-6-21 22:27

声明一下:本人看贴和回贴的规则,好贴必看,精华贴必回。  
作者: xuguofeng    时间: 2018-6-23 18:57

设置阅读啊  
作者: 加菲猫    时间: 2018-7-23 21:18

我的啦嘿嘿  




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