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【摘要】
Objective- SR-BI/apolipoprotein (apo) E double knockout (dKO) mice exhibit many features of human coronary heart disease (CHD), including occlusive coronary atherosclerosis, cardiac hypertrophy, myocardial infarctions, and premature death. Here we determined the effects on this pathology of hepatic lipase (HL) deficiency, which has been shown to significantly modulate atherosclerosis.
Method and Results- The SR-BI/apoE/HL triple knockout (tKO) mice generated for this study lived significantly longer (37%) than corresponding dKO controls (average lifespans: 63.0±0.8 versus 46.0±0.3 days), despite their increased plasma cholesterol levels. At 6 weeks of age, compared with dKO mice, tKOs exhibited significantly less aortic root and coronary artery occlusive atherosclerosis, and improved cardiac structure and function. However, by 9 weeks of age the hearts of tKO mice exhibited lipid-rich coronary occlusions, myocardial infarctions, and cardiac dysfunction essentially identical to that of 6-week-old dKO mice.
Conclusions- HL-deficiency delays the onset and/or progression of atherosclerosis via a SR-BI-independent mechanism. Extent of occlusive coronary arterial lesions was more closely associated with cardiac dysfunction and lifespan than the amount of aortic root atherosclerosis, suggesting that these occlusions in dKO mice are responsible for ischemia, myocardial infarctions, and premature death.
SR-BI/apoE double knockout (dKO) mice exhibit occlusive atherosclerotic coronary heart disease (CHD) characterized by myocardial infarctions, cardiac dysfunction, and premature death. Analysis of SR-BI/apoE/hepatic lipase (HL) triple knockout mice demonstrated that HL-deficiency reduces atherosclerosis, improves cardiac structure/function, and extends lifespan in this CHD model.
【关键词】 atherosclerosis hepatic lipase high density lipoprotein receptor myocardial infarction
Introduction
Though apolipoprotein E (apoE) or low-density lipoprotein receptor (LDLR) knockout (KO) murine models of dyslipidemia are often used to study lipoprotein metabolism and atherosclerosis, 1 they usually do not exhibit spontaneous occlusive coronary artery disease, myocardial infarction (MI), cardiac dysfunction and premature death, hallmarks of human coronary heart disease (CHD). Double knockout (dKO) mice deficient in the high-density lipoprotein (HDL) receptor (scavenger receptor class B type I, SR-BI) and apoE exhibit extensive aortic sinus atherosclerosis (advanced plaques with fibrous caps 2 that contain macrophages [unpublished data, 2005]), occlusive coronary arterial atherosclerosis (cellular and acellular plaques containing lipid , collagen, and fibrin deposits 3 ), and acute CHD when young (4 to 6 weeks old). 2-4 At 6 weeks of age, dKO hearts exhibit multiple, large infarctions with extensive fibrosis around the ventricular outflow tract and patchy MIs in the apex, right ventricular wall and interventricular septum. 3 In addition, they are hypertrophic with LV dilation, and exhibit severe dysfunction, including multiple electrocardiographic (ECG) abnormalities (ST elevation and depression, anesthesia induced conductance abnormalities (eg, bradyarrhythmias, AV blocks)), a 70% reduction in ±dP/dT, and 50% reduced ejection fraction. The dKO mice die between 5 and 8 weeks of age (mean 6 weeks). 2,3 Similarities between dKO and human CHD raised the possibility that these mice may help to study the pathophysiology of CHD and to develop genetic, pharmacological, and environmental approaches for prevention and treatment.
Hepatic lipase (HL) hydrolyzes triglycerides and phospholipids and is involved in processing chylomicron remnants, intermediate-density lipoprotein and HDL. 5 HL is primarily synthesized and secreted by the liver and is found in steroidogenic tissues. 6 HL participates in conversion of intermediate-density lipoprotein to LDL and large lipid-rich HDL to smaller HDL, thereby modulating their relative plasma distributions. 7-11 In addition to lipolytic activities, HL has ligand-binding activity and may mediate interactions of lipoproteins with cell surface proteoglycans and receptors, such as SR-BI and LDL receptor-related protein, thus facilitating endocytosis and/or selective lipoprotein lipid uptake. 12-16
Many studies have demonstrated that hepatic lipase can influence atherosclerosis, although mechanisms through which this occurs are poorly understood. Increased HL activity has been linked to formation of small, dense pro-atherogenic LDL particles in humans. 7 Conversely, reduced HL activity increases plasma HDL cholesterol levels in both humans (congenital deficiencies) and rodents (anti-HL antibodies or HL KO mice). 17,18 ApoE/HL double KO mice exhibit significantly smaller aortic root atherosclerotic lesions than apoE single KO mice, despite an increase in plasma total and very-low-density lipoprotein cholesterol 19 and altered lipoprotein structure and composition. 20 There are also studies showing that HL can be anti-atherogenic. 30,33 Thus, whereas HL modulates atherosclerosis, its precise effects vary depending on the system under study.
Here, we examined the role of atherosclerosis in CHD in dKO mice by examining the effects of HL deficiency. We found that HL deficiency significantly reduced aortic root and occlusive coronary arterial atherosclerosis, delayed the onset and/or progression of CHD, and increased longevity (37%). Our results support the proposal that occlusive coronary atherosclerosis is the likely cause of MI, cardiac dysfunction, and premature death in dKO mice. In addition, our study demonstrates that SR-BI is not essential for HL?s influence on atherogenesis.
Methods
SR-BI(-/-)/apoE(-/-)/HL(-/-) triple knockout (tKO) mice and control SR-BI(-/-)/apoE(-/-)/HL(+/+) dKO mice were generated on a 75:25 C57BL/6:SV129 background by crossing SR-BI(+/-)/apoE(-/-) females 2 with SR-BI(-/-)/HL(-/-) males. The offspring SR-BI(+/-)/apoE(+/-)/HL(+/-) females were then crossed to sibling SR-BI(-/-)/apoE(+/-)/HL(+/-) males to generate littermate tKO and dKO mice as well as breeder mice that were used to maintain the colonies. Animals were fed standard chow (Prolab 3000; PMI Feeds, St. Louis, Mo) and housed as previously described 21 and experiments followed MIT Animal Care guidelines. No significant differences were observed between males and females. Genotypes were determined by polymerase chain reaction (PCR). 2,22
All morphological, histochemical, biochemical, and cardiac functional analyses were performed as described previously 2-4,20-23 or in the online data supplement (see http://atvb.ahajournals.org).
Results
To assess the effects of inactivation of the HL gene on CHD in SR-BI/apoE dKO mice, we generated from common precursor animals 2 new strains on genetically similar mixed 75:25 C57BL/6:SV129 backgrounds: SR-BI/apoE/HL tKO and control SR-BI/apoE dKO mice. The effect of HL gene inactivation on lifespan of dKOs is shown in Figure 1 (dKO, black curve, tKO, gray). The tKOs survived 37% longer than dKOs (mean lifespans (days): dKO, 46.0±0.3 (6.6 weeks, n=160); tKO, 63.0±0.8 (9 weeks, n=94) ( P <0.0001 Logrank test). We also compared the lifespans of sibling dKOs and tKOs generated by intercrossing SR-BI(+/-)/apoE(-/-)/HL(+/-) mice. These tKO mice lived 12 to 37 days (25% to 84%) longer than their dKO siblings [n(tKO)=15, n(dKO)=16]. In both strains, a small fraction of mice (dKOs, 4.8%; tKOs, 10.5%) died within 10 days of weaning, possibly because of hypersensitivity to the anesthetic 3 used during sampling for genotyping or as yet unidentified relatively uncommon genetic or environmental factors. These animals are included in Figure 1, but not in the calculation of mean lifespans.
Figure 1. Effect of HL deficiency on survival of dKO mice. Survival curves for tKO (gray line) and dKO (black line) mice. Mice that died at 33 days of age or younger are included in the graph but were not included in the calculation of mean lifespan.
At 6 weeks of age, dKOs appeared hunched and sickly (eg, lethargy) with ruffled fur, whereas tKOs were healthier (sleeker fur, more active). Moreover, at this age tKOs weighed significantly more than dKOs (18.5±0.4 versus 16.4±0.3 gm). By 9 weeks the tKOs had grown larger (20.9±0.7 g), but otherwise resemble the ill 6-week-old dKOs. To investigate mechanisms underlying the extended lifespan of the tKOs, we further characterized the mice at &6 weeks (37 to 48 days) of age, designated dKO-6 and tKO-6 and & 9 weeks (60 to 68 days, tKO-9). As previously reported, by 6 weeks of age &50% of the dKO mice died and the surviving animals exhibited occlusive atherosclerosis, MI, and heart dysfunction, 3,4,21 whereas virtually all of the tKO mice were alive. We expected that surviving tKO-9 mice (mean age of death) might resemble dKO-6 mice.
Cardiac Function and Structure
As previously reported, 3 dKO-6s exhibited a variety of electrocardiographic (ECG) abnormalities associated with CHD, including ST depression and ST elevation indicative of ischemia and myocardial infarction 3 ( Figure 2 A). In contrast, the ECGs of tKO-6s were normal ( Figure 2 B). However, as the tKOs approached 9 weeks of age, they exhibited ECG abnormalities similar to those of dKO-6s (eg, ST depression and elevation; Figure 2 C), suggesting that, as is the case for dKOs, CHD may be the primary cause of premature death. Thus, inactivation of HL may have slowed the initiation and/or progression of CHD. The gross characteristics of the hearts support this suggestion. Hearts from tKO-6 mice were similar in surface appearance to CHD-free control hearts ( Figure 3A and 3 C), whereas those from dKO-6s and tKO-9s were markedly enlarged and exhibited surface patches characteristic of large MIs 3 ( Figure 3B and 3 D). Although the heart-to-body weight ratio for tKO-6s was significantly larger than that for controls (1.3-fold), indicating that the tKO-6 hearts were not normal; this cardiomegaly, caused at least in part by hypertrophy, 23 was significantly less than that of dKO-6s (1.9-fold) or tKO-9s (1.7-fold) ( Figure 3 E).
Figure 2. ECG analysis of cardiac function. Representative ECGs (n=4) from unanesthetized dKO-6 (A; ST depression and elevation), tKO-6 (B; normal ECG) and tKO-9 (C; ST depression and elevation) mice.
Figure 3. Anatomic and gravimetric analyses of hearts from 6-week-old SR-BI(+/-)/apoE(-/-)/HL(-/-) control, dKO, and tKO, and 9-week-old tKO mice. Intact hearts were photographed (A to D); and weighed (E; values represent mean±SEM of heart to body weight ratios; all pairwise comparisons P <0.0001). The values (mg/g) were: control, 4.85±0.12, n=20; dKO-6, 9.39±0.19, n=62; tKO-6, 6.28±0.12, n=47; and tKO-9, 8.20±0.29, n=39.
Masson?s trichrome 3 stained longitudinal heart sections ( Figure 4A to 4 C, healthy myocardium stains red, fibrotic tissue blue) showed that, unlike the massive fibrosis/MI present in virtually all dKO-6 and tKO-9 hearts ( Figures 4A and 4 C), with especially marked left ventricular dilation and fibrosis in the outflow tracts of the most ill tKO-9s, tKO-6 hearts were relatively healthy, with little fibrosis ( Figure 4 B) or intramyocardial neutral lipid deposition 3 (Oil Red O staining, data not shown). Thus, tKO mice develop MIs, cardiomegaly, left ventricular dilation, and cardiac dysfunction (ECGs) similar to those of dKO mice, but onset and/or progression of disease is slower in tKOs.
Figure 4. Histological analysis of hearts. A to C, Longitudinal Masson?s trichrome-stained heart sections (healthy myocardium, red; fibrotic tissue, blue) from 40-day-old dKO (A), 42-day-old tKO (B), and 60-day-old tKO (C) mice. D to F, Oil Red O-stained coronary artery sections (neutral lipid red) from 45-day-old dKO (D), 44-day-old tKO (E), and 68-day-old tKO (F) mice. G to I, Oil Red O-stained aortic root sections from 43-day-old dKO (G), 44-day-old tKO (H), and 62-day-old tKO (I) mice. Bars=1 mm (A,B,C), 40 µm (D,E,F), or 200 µm (G,H,I). J, Cross-sectional areas of Oil red O-stained lesions in the aortic root region. Average lesion areas (mm 2 ±SEM, horizontal lines): dKO-6, 0.148±0.019; tKO-6, 0.053±0.005; and tKO-9, 0.370±0.034. All pair-wise comparisons had P 0.0001. K, Extent of coronary arterial occlusions. The percent of essentially unoccluded (white bars), partially ( 50%; black bars) occluded coronary vessels in dKO-6, tKO-6, and tKO-9 mice.
Effects of Hepatic Lipase Deficiency on Aortic Root and Occlusive Coronary Arterial Atherosclerosis
To determine whether extended lifespan and improved cardiac pathology of HL-deficient tKO-6 mice was associated with reduced atherosclerosis, we quantified aortic root and occlusive coronary arterial atherosclerosis. Figure 4 shows representative images of Oil Red O-stained aortic roots ( Figure 4G to 4 I) and quantitative analysis of lesion sizes ( Figure 4 J). Compared with lesion areas in dKO-6s, the plaques were 3-fold smaller in tKO-6 ( Figure 4 H) and 2.5-fold larger in tKO-9 ( Figure 4 I) mice.
Comparable results were observed in coronary arteries. Vessels were scored in at least 5 Oil Red O-stained sections per mouse 50%) occluded, partially (<50%) occluded, and open (no visible plaque). In dKO-6 mice ( Figure 4D and 4 K), severe and partially occluded lesions were common (36±4%, 28±3%, respectively, n=5 mice), whereas 36±3% of the vessels were open. Occlusions, which were either predominantly acellular or contained significant cellular components, 3,21 were prevalent in areas with myocardial fibrosis, especially near the upper ventricular outflow regions. Unlike the dKO-6s, most vessels in tKOs-6s ( Figure 4E and 4 K, n=5) were completely open (62±9%), with 31±8% partially and only 6±3% severely occluded. Thus, at 6 weeks of age blood flow and oxygenation of the cardiac muscle in tKO-6s would have been better than in dKO-6s. However, in tKO-9s (n=5 mice), 46±8% of the arteries were severely occluded, 19±4% partially occluded, and only 35±9% open ( Figure 4F and 4 K), values comparable to those in dKO-6s. The extents of aortic root and occlusive coronary arterial atherosclerosis were grossly correlated with the extent of MI, cardiomegaly, cardiac function, and lifespan, suggesting atherosclerosis is most likely responsible for CHD and premature death in dKO mice and that HL-deficiency may extend life by slowing the onset and/or progression of atherogenesis. For tKO-9 and dKO-6 mice, there was a substantially larger difference in aortic root atherosclerosis (&3-fold) than in the number of occluded coronary vessels (1.02-fold), even though sampling occurred at their respective mean ages of death (9 or 6 weeks). Occlusive coronary arterial atherosclerosis is expected to cause myocardial ischemia and consequently MI, cardiac dysfunction, and death, whereas previous studies have shown that extensive aortic root atherosclerosis alone is not usually associated with MI and overt cardiac dysfunction. 24-26 Thus, the closer correlation of occlusive arterial than aortic root atherosclerosis with the cardiac phenotypes is not surprising.
Effects of Hepatic Lipase Deficiency on Lipoprotein Abundance and Composition
Figure 5 and the Table show that HL deficiency had little influence on the size distribution of the lipoproteins in dKOs and only a small effect on relative lipid compositions. At 6 weeks of age the fasting plasma total cholesterol level in tKOs was &1.6-fold higher than in dKOs, a difference caused primarily by increases in the very-low-density lipoprotein size range ( Figure 5 ); however, by 9 weeks of age the plasma total cholesterol in tKOs decreased to that in dKO-6s. Nonfasting (Table I, available online at http://atvb.ahajournals.org) and fasting plasma lipid levels were similar. Based on comparisons with healthy SR-BI(+/-)/apoE(-/-)/HL(-/-) littermates of tKO mice at 6 (n=5, TC=745±58) and 9 (n=7, TC=817±90) weeks of age, the decrease in plasma cholesterol between 6 and 9 weeks in tKO mice was not simply attributable to normal aging and/or sexual maturation. The mechanisms underlying this reduction are unclear, although it is noteworthy that higher lipoprotein levels may be cardioprotective, possibly by limiting inflammation, and reduced lipid levels may be associated with a poor prognosis in chronic heart failure. 27,28
Figure 5. Lipoprotein cholesterol profiles from mice. Plasma was size fractionated (Superose 6-fast protein liquid chromatography ) and total cholesterol in each fraction (mg/dL plasma) determined. Chromatograms shown are from 42-day-old dKO (black circles) and tKO (white circles) mice, and 62-day-old tKO mice (gray circles) and are representative of multiple independent determinations.
TABLE 1. Fasting Plasma Lipid Data From dKO-6, tKO-6, and tKO-9 Mice
We observed no significant differences in the fasting triglyceride levels of tKO-6 and dKO-6 mice or the HDL cholesterol levels of dKO-6, tKO-6, and tKO-9 mice ( Table 19,20 ). Though triglyceride levels were similar in dKO-6 and tKO-6 mice, they were higher in tKO-9 mice. The ratios of surface (phospholipids plus unesterified cholesterol) to core (cholesterol ester plus triglyceride) lipids were similar for dKO-6s and tKO-9s and significantly higher than in tKO-6s ( Table ). As previously reported, higher lipoprotein surface to core lipid ratios are associated with greater atherogenic potential in different SR-BI-deficient models of CHD. 4,23 Thus, we did not observe substantial HL-dependent differences in the lipoproteins. Further studies are necessary to determine whether subtle differences in lipoprotein structures caused by HL deficiency, or if HL-dependent changes in lipoprotein metabolism distinct from changes in particle structure (eg, as a consequence of the lipoprotein binding activity of HL), 29 were responsible for the striking effects on atherosclerosis in dKO mice.
Discussion
The SR-BI/apoE dKO mouse exhibits many hallmarks of human CHD (see Introduction). 3 It and its variant, the SR-BI KO/ ApoeR61 h/h mouse, 23 provide uniquely powerful tools to investigate the mechanisms underlying CHD and to identify potential therapeutic targets and approaches. For their promise to be realized, it is necessary to determine whether the CHD etiology and pathology in these models are similar to those in humans. After initial characterization of dKO mice, 2,3 there appeared to be two potential causes of MIs, cardiac dysfunction, and premature death: B- and T-cell-mediated inflammatory heart disease or ischemic disease caused by occlusive atherosclerosis. Previous studies, in which B- and T-lymphocyte production was blocked by deficiency in the RAG2 gene, demonstrated that B- and T-lymphocyte-mediated inflammatory heart disease is not essential for CHD in dKO mice. 21
In an attempt to directly test the role of atherosclerosis in this CHD, we generated SR-BI/apoE/HL tKO mice with the goal of changing the rate of onset or progression of atherosclerosis. Numerous reports indicate that HL can have either pro-atherogenic or anti-atherogenic activities in humans and mice, depending on the details of the system undergoing study. 30,31,32 For example, alterations in HL expression result in strikingly different effects in apoE KO and LDLR KO mice, despite the similarities in morphology of their aortic atherosclerotic lesions. HL deficiency dramatically reduces aortic root atherosclerosis in apoE knockout mice 19 via poorly understood mechanisms that likely include alterations in lipoprotein metabolism, indicating a pro-atherogenic role for HL. In contrast, hepatic overexpression of human HL in LDLR/HL double KO mice reduces aortic atherosclerosis, indicating an anti-atherogenic activity of overexpression. 33 Unlike the dKO mice examined here, these and other murine models do not exhibit significant occlusive coronary arterial atherosclerosis. Thus, the influence of HL on murine atherosclerosis of this sort has not be defined previously; however, examination of HL effects on coronary arterial atherosclerosis in humans has been reported. 30
The diverse effects of HL on atherosclerosis in different systems is not surprising, because HL is a complex protein that exhibits distinct lipolytic 16 and ligand-binding 13-15,38-41 activities that have multiple effects on lipoprotein composition and metabolism, and thus could influence atherogenesis via a variety of mechanisms. 29,33 For example, in apoE KO mice, HL deficiency may be anti-atherogenic because it raises HDL cholesterol, increases the capacity of HDL to promote cellular cholesterol efflux in vitro, 19 prevents HL-mediated increases in the atherogenicity of very-low-density lipoprotein particles, 19,34,35 increases plasma apoA-I and apoA-IV levels, 19,20 or alters the phospholipid composition of lipoproteins. 20,36,37 In addition, expression of catalytically inactive HL has been shown to produce a dramatic reduction of atherosclerosis in apoE/HL double KO mice, 29 but not in LDLR/HL double KO mice. 33 Furthermore, HL might influence atherosclerosis independently of its effects on systemic lipoprotein metabolism. For example, bone marrow transplantation experiments demonstrate localized HL expression by macrophages can dramatically influence aortic atherosclerosis. 9,42 Thus, the effects of HL deficiency on aortic root, and particularly on coronary arterial, atherosclerosis in dKO mice could not be known a priori.
HL deficiency significantly reduced both aortic root and coronary arterial occlusive atherosclerosis in dKO mice, with 64% and 42% reductions, respectively, at 6 weeks of age. Despite the striking effects of HL deficiency on atherosclerosis, we did not observe alterations in relative cholesterol and phospholipid levels in lipoproteins or the size distribution of lipoproteins, although as expected HL deficiency was accompanied by increased plasma total cholesterol. Thus, as in other model systems, the precise mechanisms through which HL deficiency reduces atherosclerosis in dKO mice remain unclear. Future studies will be required to explore the possibility that in dKO mice HL deficiency resulted in subtle, yet functionally important, changes in lipoprotein structure, binding to tissues, or metabolism.
As a consequence we could examine the effects of the reduced atherosclerosis on CHD. At 6 weeks of age, the reduced atherosclerosis in tKO mice was associated with a dramatic reduction in hypertrophy and almost complete prevention of MI, and electrocardiographically determined cardiac dysfunction (eg, ST elevation and depression). Furthermore, in tKO mice there was an increase in the mean age of death from &6 to 9 weeks. By 9 weeks of age, occlusive coronary arterial atherosclerosis in tKO mice was virtually identical to that in 6-week-old dKO mice (65% versus 64% of arteries with occlusions) and there was 1.5-fold greater aortic root disease. The abnormal cardiac phenotypes in 9-week-old tKO mice resembled those of 6 week-old dKOs. Thus, the extent of occlusive coronary arterial atherosclerosis appeared to be somewhat more closely correlated with cardiac pathology than the relative amounts of aortic root atherosclerosis.
The current study demonstrates that HL deficiency can delay the onset and/or reduce the rate of progression of atherosclerosis in the absence of SR-BI, and it seems likely that this reduction in occlusive coronary arterial atherosclerosis was responsible for the slower development of CHD and the extension of lifespan. Two other studies support this conclusion. First, the hypolipidemia and anti-atherosclerosis drug probucol dramatically blocks in dKO mice the onset and/or progression of atherosclerosis and cardiac pathology, and increases their lifespans (mean age of death increases to 36 weeks). 4 However, it is difficult to draw definitive conclusions about the role of lipoprotein metabolism and atherosclerosis in the CHD in dKO mice solely from the beneficial effects of probucol, because this drug exhibits multiple, pleiotropic activities (anti-oxidant, anti-inflammatory, cardioprotective in the absence of dyslipidemia). 43-45 Second, a lipid-rich atherogenic diet can induce in SR-BI KO/ ApoeR61 h/h mice fatal CHD that is remarkably similar to that in chow-fed dKO mice. SR-BI KO/ ApoeR61 h/h mice have low, rather than no, plasma apoE, rendering them susceptible to diet-induced hyperlipidemia, atherosclerosis and CHD. 23,46
Taken together with our earlier studies, 2-4,21 this work supports the suggestion that occlusive coronary atherosclerosis is directly responsible for ischemia-induced myocardial infarction, which in turn leads to cardiac dysfunction and premature death, a pathologic process closely resembling that in human CHD. Because rapid onset occlusive coronary arterial atherosclerosis, (to date, occlusive thrombi have not been observed in these mice [unpublished data, 2005]), associated MI does not usually accompany the aortic atherosclerosis seen in other common murine models of hyperlipidemia, 24,47-51 the dKO mouse, and its variant, SR-BI KO/ ApoeR61 h/h mice, 23 provide attractive small animal models of human occlusive atherosclerotic CHD for genetic and pharmacological studies of the mechanisms underlying the most common causes of heart disease and preclinical testing of new therapeutic strategies.
Acknowledgments
This work was supported by National Institutes of Health grants HL66105 and HL64737 (M.K.). We thank E. Vasile, S. Zhang, A. Yesilaltay, K. Makikallio, Y. Zhu, N. Liu, M. Oefinger, and S. Vassallo for assistance and discussions.
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作者单位：Department of Biology, Massachusetts Institute of Technology, Cambridge, Mass. Current address for B.T.: Department of Biochemistry and Biomedical Sciences, McMaster University. Hamilton, Canada.