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【摘要】 The effects of adriamycin on the contractile function of cultured mesangial cells were measured by the changes in planar surface area in response to treatment with agonists. Incubation of mesangial cells with adriamycin (0.2 µg/ml) for 24 h significantly decreased the contractile responses to the calcium channel activator BAY K 8644 (1 µM) and to the PKC activator PMA (1 µM). Intracellular calcium concentration ([Ca 2+ ] i ), measured by changes in fura 2 levels in response to ATP (0.1 mM), was significantly inhibited in adriamycin-treated mesangial cells compared with control cells. In the absence of extracellular calcium, treatment with ionomycin (0.1 mM) or thapsigargin (10 µM) resulted in a significantly smaller increase in [Ca 2+ ] i in adriamycin-treated mesangial cells compared with control, suggesting an important role of the endoplasmic reticulum in the effects of adriamycin. Using PKC-specific antibodies, adriamycin significantly decreased the cytosolic and membranous fractions of PKC- in mesangial cells to 75 ± 6 and 70 ± 12% of control, respectively. The PKC activity of mesangial cells was also significantly inhibited after incubation with adriamycin for 24 h. In conclusion, adriamycin induces hypocontractility of mesangial cells, which may mediate this effect by inhibiting PKC- and [Ca 2+ ] i.
【关键词】 planar surface area protein kinase C protein kinase C activity fura
A WELL - DOCUMENTED EXPERIMENTAL model of focal segmental glomerular sclerosis (FSGS) is induced by a single intravenous injection of adriamycin in rats and results in heavy proteinuria, glomerular capillary wall changes, and intraglomerular ultrafiltration very similar to those seen in FSGS ( 5, 12, 20, 26 ).
Numerous studies have demonstrated that mesangial cells are contractile and resemble vascular smooth muscle cells with respect to their signaling and cytoskeletal responsiveness to hormones ( 10, 15, 17 ). Previous studies demonstrated that the contractile function of mesangial cells regulates glomerular capillary surface area, ultrafiltration coefficient, and filtration rate ( 1, 31 ). The contraction of mesangial cells involves the interplay of many signal transduction pathways, including both PKC and intracellular calcium concentration ([Ca 2+ ] i ) ( 10, 22 ). The role of PKC and [Ca 2+ ] i in the effects of adriamycin on contractile function of mesangial cells is currently unclear.
In adriamycin nephrotoxicity, a pathological change in glomeruli is initially manifested by glomerular hypertension and hyperfiltration ( 5, 12, 26 ). This hyperfiltration of glomerular function could be due to impairment of the contractile function of mesangial cells. To test this hypothesis, we studied the effects of adriamycin on the contractile function of cultured mesangial cells. Our results demonstrated for the first time that adriamycin induces hypocontractility of mesangial cells mediated by inhibition of PKC and [Ca 2+ ] i.
MATERIALS AND METHODS
Cell culture. Mycoplasma-free SV40 MES 13 (murine) mesangial cells were obtained from the American Type Culture Collection (CRL-1927, Rockville, MD) at passage 27 and were routinely maintained in a 3:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium supplemented with 5% fetal bovine serum and 14 mM HEPES as previously described ( 24 ). Cell monolayers were routinely grown to confluence at 37°C in 5% CO 2 before the experiments, and all experiments were performed between passages 30 and 35 to minimize the effects of phenotypic variation in continuous culture.
Measurements of mesangial cell planar surface area. Cultured mesangial cells (1,000 cells/well) were seeded onto plastic 24-well cultures 2 days before the experiments. Twenty-four hours before the contractility experiments, mesangial cells were cultured with or without adriamycin (0.2 µg/ml) for 24 h.
On the day of the experiment, the 24-well dish was mounted on the heated stage of an inverted light microscope. Using a video camera attached to the microscope, 1-2 cell images/experiment were captured and stored on the hard disk of a microcomputer. The change in mesangial cell planar surface area in response to PMA (1 µM), angiotensin II (1 µM), or a calcium channel activator, BAY K 8644 (1 µM), was observed at 30°C, pH 7.4, 95% O 2 -5% CO 2. In some experiments, mesangial cells were pretreated with the PKC inhibitor chelerythrine (0.1 µM) before the contractile study. Images of the same cell were digitized serially at 5-min intervals. The perimeter of the individual cell with clearly defined borders was outlined, and the planar surface area was automatically calculated using WinLab software (DR Instruments, Taipei, Taiwan).
The change in planar surface area compared with its original size was calculated and expressed as a percentage of the initial value for each cell. The planar surface area, expressed as means ± SE, was determined for each time point.
Pharmacological agents including PMA, ATP, bradykinin, BAY K8644, angiotensin II, chelerythrine, thapsigargin, and ionomycin were purchased from Sigma (St. Louis, MO).
[Ca 2+ ] i measurements. [Ca 2+ ] i was measured by the ratiometric method in a fura 2-loaded single cell as described previously ( 6 ). After two washes with a loading buffer consisting of (in mM) 150 NaCl, 5 KCl, 5 glucose, 1 MgCl 2, 2.2 CaCl 2, and 10 HEPES, pH 7.4, cultured mesangial cells were incubated with 5 µM fura 2-AM in the same buffer at 37°C for 20 min on a coverslip. Then, the fura 2 was removed by washing, and the cells were incubated at 37°C for 10 min to convert the fura 2-ester to the free acid form under the action of nonselective esterase. The coverslip was then mounted in a modified Cunningham chamber ( 9 ) attached to the stage of an Olympus IX70 inverted microscope. The fluorescence of the fura 2-loaded cells was monitored using a dual-excitation spectrofluorometer with a photomultiplier-based detection system (Merlin, Life Science Resources) coupled to the microscope through a fiber-optic cable. Mesangial cells were excited alternatively with 340- and 380-nm light, and the emitted fluorescent light was collected. The fluorescence ratio obtained at 340 and 380 nm (F340/F380) was used as an index of [Ca 2+ ] i. A pharmacological agent (including ATP or bradykinin) was administered by adding the solution to one side of the Cunningham chamber and draining it through the other side with filter paper. [Ca 2+ ] i was monitored immediately thereafter. [Ca 2+ ] i of most mesangial cells peaked 5 min after the addition of the pharmacological agent. Some experiments were performed in calcium-free conditions, with calcium being omitted from the loading buffer and 0.5 mM EGTA added during [Ca 2+ ] i measurement. All experiments were performed using at least 18 cells. The results were expressed as means ± SE for the ratio increase.
Cell fractionation and PKC immunoblotting. PKC isoforms were probed in cytosolic, membranous, and particulate fractions of growth-arrested mesangial cells after incubation with or without adriamycin (0.2 µg/ml) for 24 h. Cells were washed twice with ice-cold PBS and harvested in 100 µl/100-mm plates with buffer A containing (in mM) 1 NaHCO 3, 5 MgCl 2 -6H 2 O, 50 Tris·HCl, 10 EGTA, 2 EDTA, 1 DTT, and 1 phenylmethylsulfonyl fluoride, as well as 25 µg/ml leupeptin and 10 µM benzamidine. The cells were passed through a 26-gauge needle three times and incubated on ice for 30 min. Homogenates were centrifuged at 100,000 g for 1 h at 4°C, and the supernatants were retained as the cytosolic fraction. The pellet was dissolved in 100 µl of buffer B ( buffer A with 1% Triton X-100), passed through a 26-gauge needle, and centrifuged again for 1 h at 100,000 g at 4°C. The supernatant was collected and used as the membranous fraction. The remaining Triton X-insoluble pellet was solubilized in 100 µl of 10% SDS, boiled for 10 min, and served as the particulate fraction. Protein concentration was determined using a modified Lowry microassay (Bio-Rad, Hercules, CA). Aliquots were then denatured in 4 x SDS sample buffer, boiled for 5 min, and loaded onto 10% polyacrylamide gels. Samples were electrophoresed at 45 V for 20 min, then at 100 V for 60 min at room temperature. After 3 h of transfer (225 mA), the polyvinylidene difluoride membrane was blocked overnight at room temperature in PBS buffer containing 10% nonfat milk. The membrane was then exposed to monoclonal anti-PKC- (1:250), - (1:250), or - (1:1,000) antibodies (BD Biosciences) for 1 h, followed by a 1-h incubation with a horseradish peroxidase-conjugated affinity-purified goat anti-mouse IgG antibody (BD Biosciences) diluted at 1:1,000. The blots were rinsed in PBS buffer between each of the preceding steps. The secondary antibody was detected by enhanced chemiluminescence, and the membrane was developed on Kodak Biomax film. Densitometry was performed to analyze the relative density of the protein bands.
PKC activity assay. Mesangial cells (1 x 10 7 cells) treated with or without adriamycin (0.2 µg/ml) for 24 h were collected and sonicated in ice-cold PBS. The cell lysate was centrifuged at 100,000 g for 60 min, and the supernatant was collected. Protein concentration was determined using Lowry's protocol. Kinase activity (in a 12-µl sample) was measured using a nonradioactive protein kinase assay kit (Calbiochem), and optical density values were read at 492 nm in a microplate reader ( 16 ).
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay. For 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay, mesangial cells were cultured in 96-well culture plates at a density of 1,000 cells/well with 200 µl of culture medium. After overnight plating, the adriamycin (0.2 or 2 µg/ml) was added for 24-72 h. At the time of evaluation of cell growth, 20 µl of MTT (5 mg/ml in PBS solution) were added to each well. After another 3-h incubation at 37°C, the supernatant was added to 25 µl of Sorenson's glycine buffer and 200 µl of DMSO to dissolve the blue formazan produced by the mitochondrial succinate dehydrogenase of living cultured mesangial cells. Absorbances were measured by a spectrophotometer at a test wavelength of 540 nm ( 18 ).
Statistical analyses. All results are expressed as means ± SE. Cultured mesangial cells were subcultured the same day for control and adriamycin-treated groups. Contractile responses, immunoblots, and PKC activity were assayed in duplicate on the same day. Statistical analysis was performed using a paired Student's t -test. A P value <0.05 was considered to be statistically significant.
RESULTS
Treatment of cultured mesangial cells with adriamycin (0.2 µg/ml) for 24 h did not significantly change the morphology of cells ( Fig. 1 ). The planar surface area of cultured mesangial cells before stimulation with the agonist was 2,435 ± 368 µm 2 for the control group and 2,408 ± 454 µm 2 for the adriamycin-treated group (data not shown).
Fig. 1. Representative cell images of cultured mesangial cells with (Adriamycin) or without (Control) the treatment of adriamycin (0.2 µg/ml) for 24 h in response to 1 µM PMA (Control+PMA; Adriamycin+PMA ) for 30 min.
By applying the PKC activator PMA (1 µM), we were able to determine whether adriamycin changes PKC-induced contraction in mesangial cells. During PMA application for 30 min, the surface area of control mesangial cells decreased to 45 ± 4% of the initial surface area. However, the planar surface area of adriamycin-treated mesangial cells in response to PMA application for 30 min was only decreased to 69 ± 7% of the initial surface area ( Figs. 1 and 2 A ). Pretreatment of mesangial cells with the PKC inhibitor chelerythrine (0.1 µM) for 24 h fully eliminated the difference in planar surface area between control and adriamycin-treated cells ( Fig. 2 B ). These results suggested that PKC has an important role in adriamycin-induced hypocontractility of cultured mesangial cells.
Fig. 2. Change in mesangial cell planar surface area in response to PMA treatment for 30 min between control ( and ) and adriamycin-incubated (0.2 µg/ml, and ) cells with ( B ) or without ( A ) the presence of the PKC inhibitor chelerythrine (0.1 µM). Values are means ± SE. *Significant difference between control and adriamycin groups ( n = 22-24, P < 0.05).
Figure 3 shows the contractile responses of control and adriamycin-treated mesangial cells to angiotensin II (1 µM) with or without the presence of the PKC inhibitor chelerythrine (0.1 µM). The change in planar surface area was not significantly different between the control and adriamycin-treated cells. Pretreatment with chelerythrine partly eliminated the contractile responses to angiotensin II.
Fig. 3. Change in mesangial cell planar surface area in response to angiotensin II (ANG II) for 10 min between control ( and ) and adriamycin-incubated (0.2 µg/ml, and ) cells with ( B ) or without ( A ) the presence of the PKC inhibitor chelerythrine (0.1 µM). Values are means ± SE of 26-28 cells.
To determine whether adriamycin alters the calcium-dependent contractile responses, the contraction of mesangial cells was stimulated with the calcium channel activator BAY K 8644 (1 µM). Figure 4 shows the change in planar surface area of mesangial cells in response to BAY K 8644 expressed as a percentage of the time 0 value. During stimulation with BAY K 8644, the surface area of control mesangial cells decreased over 30 min to 66 ± 4% of the initial area. In comparison, the contractile responses to BAY K 8644 of adriamycin-treated mesangial cells were significantly blunted (planar surface area only decreased to 95 ± 9% of the initial area), suggesting that adriamycin may inhibit the process of calcium activation.
Fig. 4. Change in mesangial cell planar surface area in response to BAY K 8644 (1 µM) treatment for 30 min between control ( ) and adriamycin-incubated (0.2 µg/ml, ) mesangial cells. Values are means ± SE. *Significant difference between control and adriamycin groups ( n = 18, P < 0.05).
Immunoblotting of mesangial cells with PKC isoforms (PKC-, -, -, -, -, -, -, or - ) showed that cultured mesangial cells only express PKC-, -, and - at detectable levels ( Fig. 5 ). Figure 6 illustrates the particulate, cytosolic, and membranous expression of PKC isoforms in control and adriamycin-treated mesangial cells. Adriamycin significantly decreased PKC- content of cytosolic and membranous fractions to 75 ± 6 and 70 ± 12% of control, respectively, without significantly changing particulate fraction PKC-. Treatment of mesangial cells with adriamycin for 24 h significantly inhibited the particulate fraction PKC- content. However, cytosolic and membranous fraction PKC- content was not significantly altered by the treatment with adriamycin. Treatment with adriamycin for 24 h did not significantly change PKC- in the particulate, cytosolic, and membranous fractions.
Fig. 5. Representative immunoblots of mesangial cell particulate (Part), cytosolic (Cyto), and membranous (Mem) distribution of PKC-, PKC-, and PKC- in control (C) and adriamycin (A; 0.2 µg/ml for 24 h)-treated cells.
Fig. 6. Mesangial cell subcellular distribution of PKC-, PKC-, and PKC- in response to adriamycin (0.2 µg/ml). Histogram values are means ± SE of band densitometry data measured in 5 experiments. * P < 0.05 vs. control.
The relative PKC activity of the adriamycin-treated mesangial cells is illustrated in Fig. 7. Treatment of mesangial cells with adriamycin for 24 h significantly blunted the activity of PKC.
Fig. 7. Relative PKC activity in control and adriamycin (0.2 µg/ml for 24 h)-treated cells. *Significant difference between control and adriamycin groups ( P < 0.05).
To evaluate the extent to which calcium-induced contraction was impaired by adriamycin treatment, [Ca 2+ ] i, expressed as the F340/F380 ratio, was measured in fura 2-loaded mesangial cells. As shown in Table 1, in response to ATP (0.1 mM) stimulation in the presence of extracellular calcium, the increase in the F340/F380 ratio in adriamycin-treated mesangial cells was significantly less than that of control ( 1.1 ± 0.3 for control mesangial cells and 0.3 ± 0.1 for adriamycin-treated mesangial cells). There was no significant difference in the F340/F380 ratio in response to bradykinin (10 µM) between control and adriamycin-treated mesangial cells. To study the role of total intracellular pools of calcium in regulating [Ca 2+ ] i of mesangial cells, ionomycin (10 µM) was applied to the cells without the presence of extracellular calcium. The maximal increase in the F340/F380 ratio in response to ionomycin was 1.8 ± 0.3 for control mesangial cells and 1.1 ± 0.2 for adriamycin-treated mesangial cells (the latter being significantly lower than that of control). Adriamycin significantly inhibited the maximal increase in the F340/F380 ratio in response to the application of thapsigargin (inositiol-3,4,5-trisphosphate-independent intracellular calcium releaser) in the absence of extracellular calcium compared with control.
Table 1. Fura 2 F340/F380 ratio in control and adriamycin-treated mesangial cells
As shown in Fig. 8, direct application of adriamycin at a high dose (2 µg/ml for 48 or 72 h) to cultured mesangial cells significantly inhibited the proliferation of cultured mesangial cells as detected by MTT. Application of adriamycin at a low dose (0.2 µg/ml) did not significantly alter the growth curve of mesangial cells for up to 48 h. However, application of adriamycin at a low dose (0.2 µg/ml) for 72 h did significantly inhibit the proliferation of mesangial cells. This result suggests that the low dosage (0.2 µg/ml) of adriamycin used in this study had no cytotoxic effects on mesangial cells for up to 48 h.
Fig. 8. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay of cultured mesangial cells incubated in control or adriamycin (0.2 or 2 µg/ml) for up to 72 h. Six different cell cultures were performed as described in MATERIALS AND METHODS. Values are means ± SE. *Significant difference between control and adriamycin groups ( P < 0.05).
DISCUSSION
Our results clearly demonstrated that the incubation of cultured mesangial cells with adriamycin for 24 h significantly decreases the contractile responses to BAY K 8644 (calcium channel activator) or PMA (PKC activator), indicating that adriamycin could inhibit the activation of [Ca 2+ ] i and PKC. The effects of adriamycin include inhibiting intracellular calcium mobilization by ATP, decreasing the expression of cytosolic and membranous fractions of PKC-, and blunting PKC activity, resulting in the hypocontractility of adriamycin-treated mesangial cells. These results may suggest that adriamycin could influence the regulatory function of mesangial cells on the glomerular capillary surface, mediated by [Ca 2+ ] i and PKC-dependent pathways.
Increased glomerular flows and pressures have been proposed as an important pathogenetic mechanism for FSGS in the animal model ( 5, 12, 26 ). The histological lesions resulting in FSGS start in the mesangial area with segmental deposition of immunoglobulin M, complement, fibrinogen, and lipoproteins ( 8, 11 ). Although currently no direct evidence indicates that the impairment of mesangial cell contraction leads to matrix deposition in FSGS, other investigators have suggested that impairment of mesangial cell function might cause increased matrix substance deposition and development of FSGS ( 8, 11 ).
Morphologically and physiologically, glomerular mesangial cells are well suited to a capillary surface area-regulating function, as they possess numerous intracellular contractile myofilaments ( 7, 27 ). Evidence has now accumulated from a variety of sources suggesting that the glomerular mesangial cell may be the site of the common pathway by which many of the agonists (angiotensin II or ATP) decrease the ultrafiltration coefficient of the glomerulus by reducing glomerular capillary surface area, and hence the single-nephron glomerular filtration rate, through the contraction of mesangial cells ( 14, 25 ). Our measurements of planar surface area were consistent with previous studies that reported a mean decrease of 25-40% in mesangial cell surface area at 30 min in response to PMA ( 10, 15, 23 ). Thus adriamycin nephropathy could impair the regulatory function of mesangial cells on the glomerular capillary surface area and of the glomerular filtration coefficient as demonstrated by our contractility study.
A decrease in glomerular volume or surface area in response to angiotensin II is also seen in isolated glomeruli and mesangial cells, respectively ( 15, 22 ), presumably due to mesangial cell contraction. In our experiment, the change in planar surface area in response to angiotensin II was slightly but not significantly inhibited in adriamycin-treated mesangial cells compared with control cells. This may suggest that adriamycin has a selective effect on PMA- and BAY K 8644-induced contraction without significantly altering the physiological responses of mesangial cells to angiotensin II.
Intracellular free calcium regulates many cellular functions, including contraction. An increase in [Ca 2+ ] i from intracellular calcium stores (via inositiol-3,4,5-trisphosphate generation) and calcium influx (via receptor-operated calcium channels) occurs in response to G protein-coupled receptor agonists (ATP or bradykinin) ( 2, 4 ). The reduced magnitude of the elevation of [Ca 2+ ] i in response to ATP in adriamycin-treated mesangial cells indicates that either the dissociation of receptor-coupled calcium channels or/and the depletion of the intracellular calcium stores is responsible for adriamycin-mediated effects.
We therefore examined the amount of available calcium remaining within the intracellular calcium stores using ionomycin in the absence of extracellular calcium as an index. As shown in Table 1, the increase in the F340/F380 ratio induced by ionomycin was significantly less in the cells that had been subjected to adriamycin incubation. These results suggest that the decreased response (F340/F380 ratio) to ATP is at least partially due to the filling state of the intracellular calcium stores. To further characterize the contribution of the endoplasmic reticulum to [Ca 2+ ] i, we examined the effect of thapsigargin on [Ca 2+ ] i. Thapsigargin blocks calcium loading of the intracellular calcium stores by inhibiting the endoplasmic reticular calcium pump and depleting the calcium stores of the endoplasmic reticulum ( 4 ). As shown in Table 1, the thapsigargin-induced change in the F340/F380 ratio in mesangial cells in calcium-free buffer was significantly inhibited in adriamycin-incubated mesangial cells compared with control. Taken together, these results indicate an important contribution of the endoplasmic reticulum to the effects of adriamycin on [Ca 2+ ] i in cultured mesangial cells.
Intracellular PKC is implicated in the phosphorylation of myosin light chain, which promotes both actin-activated myosin Mg 2+ -ATPase activity and cross-bridging required for cell motility or contraction ( 28 ). The immunoblot data in this study revealed that adriamycin significantly decreases the expression of the PKC- isoform in cytosolic and membranous fractions of mesangial cells. Because PKC- is a diacylglyerol (DAG)-sensitive and Ca 2+ -dependent PKC, the decreasing expression of PKC- may contribute to the hypocontractility of adriamycin-treated mesangial cells to BAY K 8644 (calcium-dependent constrictor) or PMA (PKC-dependent constrictor). The results of measuring PKC activity (the value being adjusted to the protein concentration) in control and adriamycin-treated mesangial cells also support our hypothesis that adriamycin also inhibited the activity of PKC. Because adriamycin inhibited both the protein level and activity of PKC, the actual blunting effect of adriamycin on PKC activity was amplified in mesangial cells.
Adriamycin also significantly decreased the expression of PKC- (atypical PKC) in the particulate fraction without significantly changing the expression of cytosolic and membranous fractions of PKC-. Because PKC- is a DAG-insensitive and Ca 2+ -independent isoform, it may not have a significant role in contractile responses of mesangial cells to agonists ( 3, 19 ). Further studies are needed to determine whether adriamycin-induced decreases in the expression of particulate fraction PKC- have long-term effects on the signal transduction of mesangial cells ( 3, 19 ). Because the effect of adriamycin on the expression of PKC isoforms changed both Ca 2+ - and DAG-dependent and -independent PKC isoforms, it would be more meaningful and offer mechanistic insight by using dominant negative cDNAs to different PKC isoforms in a future study.
The pathological alterations of adriamycin-induced hypocontractility of mesangial cells could be due to the free radicals generated by adriamycin, as suggested by previous investigators who showed that adriamycin nephrotoxicity may be linked to the transfer of oxidative stress to cellular compounds facilitated by the ability of quinines to form semiquinone radicals ( 21 ). Previous studies also demonstrated tumor necrosis factor, platelet-activating factor, and interleukin-6 synthesis by mesangial cells after stimulation with adriamycin ( 13, 30 ). Thus the hypocontractility effects of adriamycin could also be due to the release of different cytokines or chemokines from mesangial cells and thereby to further impairment of the contractility of mesangial cells.
A previous study demonstrated that mesangial cell contraction occurs via different activation pathways in that phosphorylation of myosin light chain serine-1, serine-2, and threonine-9 is PKC dependent, whereas the Ca 2+ /calmodulin-dependent myosin light chain kinase phosphorylates threonine 18 and serine 19 ( 29 ). Thus adriamycin may inhibit the phosphorylation activity of Ca 2+ /calmodulin- or/and PKC-dependent myosin light chain kinase.
In conclusion, adriamycin induces hypocontractility of mesangial cells, which may mediate this effect by inhibiting PKC- and [Ca 2+ ] i. Impairment of mesangial contractile capability by adriamycin may jeopardize glomerular hemodynamics and lead to hyperfiltration and FSGS in adriamycin nephropathy.
GRANTS
This study was supported by National Science Council Grants NSC 92-2314-B-016-013, NSC 92-2320-B-016-030 and National Health Research Institute Grant NHRI-EX93-9137SN (Taiwan).
ACKNOWLEDGMENTS
The authors thank Prof. Abdalla Rifai for constructive suggestions and Hsiao-Chia Jen for excellent technical assistance.
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作者单位：1 Department of Pathology, Tri-Service General Hospital, and 2 Department of Biochemistry, National Defense Medical Center, Taipei 11 Taiwan, Republic of China