Skeletal adenylate cyclase: Effect of Mg2+, Ca2+, and PTH

Summary Plasma membranes were prepared from mineralized guinea pig bone in order to study Mg²⁺ and Ca²⁺ modulation of skeletal adenylate cyclase. Plasma membrane preparation was accomplished by crushing the bone in liquid N₂ and subsequent multiple washings in buffer containing EGTA to remove all Ca²⁺ prior to adenylate cyclase assay. Skeletal adenylate cyclase was found to be dependent on GTP and Mg²⁺ and responsive to bovine 1-34 PTH. Ca²⁺ caused a competitive inhibition of Mg²⁺-activated skeletal adenylate cyclase. The apparent KaMg was 1.9±0.3 in the presence of 0.2 μM Ca²⁺ but increased to a mean of 7.2±1.3 in the presence of 5.0 μM Ca²⁺. Analysis of the Ca²⁺ inhibition curves at concentrations from .05 μM-1.0 mM were consistent with the presence of two Ca²⁺ inhibition sites, one with an apparent Ki of 1–2 μM and the other with an apparent Ki of approximately 500 μM. Lowering the Mg²⁺ concentration increased the contribution of the high affinity Ca²⁺ binding site to the overall Ca²⁺ inhibition, and raising the Mg²⁺ concentration had the opposite effect. While bPTH 1-34 enhanced adenylate cyclase activity, it did not increase the affinity of Mg²⁺ for skeletal adenylate cyclase nor did it alter the K_iCa or the pattern of Ca²⁺ inhibition. These data may explain the skeletal resistance to PTH during Mg deficiency. A fall in the intracellular Mg would render the adenylate cyclase more susceptible to inhibition by the prevailing intracellular Ca²⁺ concentration. Since PTH does not appear to modulate either Mg²⁺ activation or Ca²⁺ inhibition of skeletal adenylate cyclase, cAMP-mediated PTH induction of bone resorption would be impaired, and hypocalcemia would occur.     

Lack of effect of ovariectomy on divalent cation regulation of skeletal adenylate cyclase

Both estrogen and androgen have been reported to attenuate cyclic AMP responses to parathyroid hormone stimulation in cultured bone cells. The present study examines the effect of in vivo estrogen deficiency on skeletal adenylate cyclase (AC) activity. The AC activity was compared in bone membranes prepared from normal female guinea pigs and from age-matched guinea pigs 3 weeks after ovariectomy. Histomorphometric analysis of femoral specimens from the ovariectomized guinea pigs demonstrated significant decreases in percentage bone volume, the percentage eroded surfaces and osteoclast numbers, and increased osteoid thickness, compared with the normal controls. No differences were found in basal AC activity, the ability of bone AC to be stimulated by parathyroid hormone (bPTH(1–34)) or isoproterenol, or in the regulation of AC activity by calcium and magnesium. We conclude that bone AC is not a direct target for estrogen effects on bone cells and that the reported effects of sex steroids on cAMP levels in bone cells probably act via an indirect mechanism.     

胆源性脓毒症导致肝细胞膜腺苷酸环化酶失敏

本研究目的是检测胆源性脓毒症期间肝细胞膜腺苷酸环化酶（ＡＣ）活性及其对激素的反应性。于免右侧叶肝管开口近端结扎肝总管，并向梗阻近端肝管内注入２１×１０８Ｅ。Ｃｏｌｉ／０．２ｍｌ大肠杆菌菌液诱发脓毒症。在伤后１２、２４和４８ｈ，参照Ｓａｌｏｍｏｎ的方法测定右侧叶（非感染肝叶）和左侧叶（感染肝叶）肝细胞膜腺苷酸环化酶（ＡＣ）活性。自病程早期始感染肝叶肝细胞膜氟化钠刺激ＡＣ活性以及肾上腺素／胰高糖素刺激ＡＣ活性和肾上腺素刺激ＡＣ活性发生先后不同程度降低。本研究结果表明，在胆源性脓毒症状态下动物感染肝叶和非感染肝叶肝细胞膜腺苷酸环化酶系统的功能完整性先后受损，这可能是肝细胞对胰高糖素和肾上腺素反应性低下的原因。     

Relative sensitivity of kidney and bone to the amino-terminal fragment b-PTH (1–30) of native bovine parathyroid hormone: Implications for assessment of bioactivity of parathyroid hormone fragments in vivo and in vitro

Summary Adenylate cyclase activation in renal cortical membranes is widely used to assess the potency of parathyroid hormone (PTH) and hormonal fragments. Recent studies, however, suggest that the structural requirements for biological activity of PTH in bone may differ from those in kidney. In isolated perfused canine bone, cyclic AMP production is markedly increased by syn-b-PTH (1–34) whereas intact b-PTH (1–84) has minimal effect. Furthermore, a potent inhibitor of PTH stimulated adenylate cyclase activity, Nle⁸Nle¹⁸Tyr³⁴ syn-b-PTH (3–34) NH₂, devoid of biological activity in kidney, is an agonist in isolated bone. The present studies examine the effects in bone and kidney of the amino-terminal fragment b-PTH (1–30), prepared by cleavage of intact b-PTH (1–84) by Cathepsin Bin vitro. In basolateral canine renal cortical membranes, b-PTH (1–30) was less active than syn-b-PTH (1–34) in its ability to stimulate adenylate cyclase. Half maximal stimulation of adenylate cyclase activity by b-PTH (1–30) was 50 nM compared with 2 nM for syn-b-PTH (1–34). Maximum enzyme activation by b-PTH (1–30) reached only 50% of the activation by syn-b-PTH (1–34). Addition of Gpp(NH)p (1 mM) increased the affinities for both peptides. The relative difference in potency, however, remained unchanged. In contrast, in isolated bone, b-PTH (1–30) and syn-b-PTH (1–34) were equipotent in increasing cyclic AMP production. These data provide further evidence for differences in the structural requirements for PTH activity in bone and kidney and suggest that bioassays of PTH and PTH fragments in kidney may not accurately reflect the effects of PTH in bone.     

Long-term administration of vitamin D3 metabolites alters PTH-responsive osteoblastic adenylate cyclase in rats

Summary We have examined the effect of induced hyper D₃ vitaminosis on bone-related variables in the rat with special reference to the parathyroid (PTH)-sensitive adenylate cyclase (AC) in rat calvariae. Subcutaneous injections three times a week of doses theoretically corresponding to about 10 times the average physiological serum levels of either 25 hydroxyvitamin D₃ (25OHD₃), 1,25 dihydroxyvitamin D₃ (1,25(OH)₂D₃), or 24,25 dihydroxyvitamin D₃ (24,25(OH)₂D₃) for 12 weeks gave the following results: At 12 weeks of treatment, 24,25(OH)₂D₃ levels in the groups receiving 25OHD₃ or 24,25(OH)₂D₃ increased significantly, whereas 1,25(OH)₂D₃ levels remained unaffected. Correspondingly, PTH-sensitive AC activities in crude calvarial membrane fractions from 25OHD₃-and 24,25(OH)₂D₃-treated animals were obliterated. This effect was apparent after 4 weeks of treatment. In the group receiving 25OHD₃, both basal, plus Gpp(NH)p-, and forskolin-sensitive AC activities were significantly reduced after 4 weeks of treatment. Similar effects in crude kidney membrane fractions were, however, not observed. Liver membranes from 25OHD₃- or 24,25(OH)₂D₃-treated animals showed insignificant changes in the isoprenalin-, PGE₁-, Gpp(NH)p-, or forskolin-sensitive AC activities. Finally, the significance of reduced PTH-sensitive bone AC activity has been assessed. 25OHD₃ treatment yielded normocalcemic and hypercalciuric rats, whereas 1,25(OH)₂D₃ enhanced both serum and urine Ca²⁺ levels. 24,25(OH)₂D₃-treated and control animals were undiscernible in this respect. However, the 24,25-(OH)₂D₃ treatment caused reductions in both serum alkaline phosphatase levels and urinary hydroxyproline/creatinine ratio. These results indicate that administration of vitamin D₃ metabolites which increase serum 24,25(OH)₂D₃ levels without affecting renal handling of Ca²⁺, obliterates the PTH-sensitive AC in bone, thereby altering bone turnover.     

Development of the cyclic AMP response to parathyroid hormone and prostaglandin E2 in the embryonic chick limb

Summary The developing chick limb was studied to determine the ability of parathyroid hormone (PTH) and prostaglandin E₂ (PGE₂) to increase intracellular cyclic AMP (cAMP) during various stages of development. All developmental stages examined (stages 20–21, 24–25, and 26–28) responded to PGE₂ when the cells were assayed immediately following the removal of the limbs from the embryos. In contrast, only stage 26–28 limb cells responded to PTH when assayed in a similar manner. The response to PTH was temporally correlated with the appearance of cartilage matrix in vivo. Undifferentiated limb cells were also cultured and assayed at various times for hormone responsiveness. Stage 24–25 high-density cell cultures responded initially to PGE₂ but not to PTH. However, by 36 h and in all subsequent itme intervals tested, the response to PTH was significantly greater than that to PGE₂. The PTH receptor, in contrast to that of PGE₂, was shown to be sensitive to trypsin treatment, but could be regenerated during subsequent cell culture. The majority of the hormoneresponsive cells were found in cartilaginous regions of the limb, and were shown to respond to both hormones in a dose-dependent manner. The PTH-induced cAMP response was affected by low cell density and mouse serum, both of which significantly inhibit the chondrogenic potential of cultured limb cells. These findings are consistent with a temporal correlation between the development of the PTH response and chondrogenesis in vivo.     

Effects of parathyroid hormone on alkaline phosphatase activity and mineralization of cultured chick embryo tibiae

Summary The objectives of this study were (a) to determine if decreased bone alkaline phosphatase (AlPase) activity, resulting either from exposure to parathyroid hormone (PTH) or from direct inhibition of AlPase with levamisole, was concomitant with net changes in bone Ca and P content; and (b) to determine the duration of the effect of PTH on bone AlPase activity after the hormone was removed. Tibiae from 7-day chick embryos were incubated in chemically defined medium for 3–4 days. The Ca and P content of bones incubated for 72 h in this system increased from 2-to 9-fold. Addition of PTH (1 U/ml) to the medium resulted in a 60% decrease in bone AlPase activity, and this decrease was accompanied by a marked inhibition of Ca and P accumulation in bone. When levamisole, a specific inhibitor of AlPase activity, was added to the medium, a similar inhibition of bone mineral accumulation resulted. A 24-h incubation in medium containing PTH (1 U/ml) resulted in a 30% decrease in bone AlPase activity. Following the 24-h exposure to PTH, incubation was continued in medium containing no hormone. After 72 h in hormone-free medium, bones that had been exposed to PTH earlier contained more AlPase activity than paired control bones never exposed to PTH. The PTH-treated bones also recovered their ability to accumulate Ca and P. The results of this study show that: (a) decreased bone AlPase activity resulting from either exposure to PTH or direct inhibition by levamisole is associated with a marked decrease in mineral accumulation in bone; (b) exposure to PTH followed by removal of the hormone leads to recovery and even an increase in bone AlPase activity; and (c) following removal of PTH from the culture system, tibiae recover their ability to accumulate Ca and P. These results suggest that one of the ways PTH may alter mineral accretion is through its effects on bone AlPase activity.     

Selective stimulation of net calcium efflux from chick embryo tibiae by parathyroid hormone in vitro

Summary The purpose of this study was to measure in an in vitro system the movement of Ca and phosphate (P_i) out of bone when treated with parathyroid hormone (PTH). Tibiae from 13-day chick embryos were incubated for up to 8 h in a defined medium containing 1.8 mM Ca. Medium samples were collected every 2 h and were analyzed for Ca, P_i and lactate. Net effluxes from the bones were calculated. When bones were incubated with PTH in the medium (1 U/ml), net Ca efflux was increased 44, 60 and 100% at 4, 6 and 8 h, respectively. At no time was net P_i efflux affected by the hormone. The well known PTH-stimulated lactate production was not seen until 8 h. Lower doses of PTH (0.1 and 0.3 U/ml) were also effective. Comparing PTH (1 U/ml) responsiveness at higher (2.2 mM) and lower (0.9 mM) medium Ca concentrations, showed that with 2.2 mM Ca no increased Ca efflux was seen, while with 0.9 mM Ca significant elevation in medium Ca occurred 2 h sooner than in the experiments using 1.8 mM Ca. In another experiment, varying the medium P_i level from 1 to 2 mM had no effect on the Ca response to PTH. In neither experiment was P_i release affected by PTH. The results of this study have led to the following conclusions: (1) PTH acts on bone to cause an early dose related increase in net Ca efflux; (2) the effect is specific for Ca, since it is not accompanied by an increased P_i efflux, and may be saturated by raising the medium Ca level; and (3) PTH-stimulated Ca efflux in this system is not correlated with, and is probably not a result of increased lactate production.     

Cerebral tumours induced by ENU; changes of adenylate cyclase activity in the tumour latency time

Summary Tumours of the nervous system have been induced by transplacental ENU. Until the fourth month of life the tumoural lesions appear as mixed glial proliferations or oligodendroglial foci. From the fourth month on they develop as glial micro- and macrotumours or as isomorphic and polymorphic oligodendrogliomas.The adenylate cyclase activity studied during these two distinct phases of tumour development was markedly reduced in brain tumours, independent of their cellular origin, compared with the level in the normal brain. On the other hand, the activity of the enzyme responsible for the synthesis of cyclic AMP is significantly increased during the first period of tumour development when early neoplastic proliferations are present.     

Stimulation of alkaline phosphatase activity in cultured neonatal mouse calvarial bone cells by parathyroid hormone

Summary The effect of parathyroid hormone (PTH) on alkaline phosphatase activity was examined in confluent, serum-free primary cultures of neonatal mouse calvarial cells. It was found that synthetic bPTH-(1-34) caused an increase in the specific activity of skeletal alkaline phosphatase isoenzyme by 18 hours. Between 10 and 500 ng/ml, the mganitude of the change was directly related to peptide concentration. The change occurred in the absence of any effect on cell number, total cell protein, or DNA and was not the result of an effect on either proliferation or survival of a specific cell population. Results of histochemical studies indicate that bPTH-(1-34) caused an increase in the proportion of cells containing enzyme activity. The response was duplicated by intact bPTH-(1-84) and DBcAMP, but not by oxidized bPTH-(1-34) or insulin and did not require prostaglandin synthesis or hydroxylation of 25-hydroxyvitamin D₃. These results demonstrate that bPTH has a direct effect on osteoblast maturationin vitro, that the effect is specific for PTH, and suggest that it is mediated by cAMP.     

甲状旁腺素对大鼠系膜细胞增殖及凋亡的影响

目的观察甲状旁腺素(PTH)对体外培养的大鼠肾小球系膜细胞(GMC)增殖及凋亡的影响,并探讨其作用的可能机制。方法体外培养的大鼠系膜细胞经不同浓度的PTH作用后,采用噻唑蓝(MTT)法检测细胞增殖,流式细胞仪测定细胞周期及凋亡,吖啶橙荧光活体染色观察细胞凋亡形态,免疫细胞化学结合计算机图文分析系统检测c-jun、c-fos表达。结果PTH持续刺激GMC6h、12h时,10-11～10-9mol/L浓度范围内,促进细胞增殖(P<0.05);10-9mol/L在12h达高峰,并可使G0～G1期细胞减少,S G2M期细胞增多,并能明显促进c-jun、c-fos的表达(P<0.01),而10-8mol/L无明显作用;刺激24h时各浓度均无作用;刺激48h各浓度明显抑制大鼠系膜细胞增殖(P<0.01),且使细胞阻滞在G0～G1期,不能进入S期,并能明显抑制c-jun、c-fos的表达(P<0.01)。PTH不诱导GMC凋亡。结论PTH体外刺激GMC增殖与作用时间及剂量相关,低浓度短时间作用刺激GMC增殖,持续作用抑制GMC增殖,其作用与c-jun、c-fos的表达有关,并影响细胞周期。PTH对体外培养的大鼠GMC凋亡无诱导作用。甲状旁腺机能亢进可能参与了慢性肾病残余肾功能进一步恶化的病理过程。     

Guanine nucleotide mediated desensitazation of adenylate cyclase in cell free preparations from a Leydig cell tumour

Cell free desensitization of a tumour Leydig cell plasma membrane adenylate cyclase has been demonstrated in the presence of guanine nucleotides. In experiments in which the membranes were pre-incubated with various nucleotides and LH, it was shown that this decreased adenylate cyclase activity was dependent on the presence of GTP and occurred both in the presence and absence of ATP. While pre-treatment with LH alone appeared to enhance subsequent adenylate cyclase activity, this hormone was able to potentiate the desensitizing effect of GTP. The desensitizing effect of GTP was not inhibited by sodium fluoride. In contrast, the GTP analogue p(NH)ppG (guanosine 5'β, λ-imido triphosphate) caused a persistent activation of the adenylate cyclase. GMP and guanosine also initially inhibited the adenylate cyclase activity, but this was entirely reversed by p(NH)ppG plus LH. GDP in addition to GTP caused desensitization but this was only partially reversed by p(NH)ppG plus LH. It is proposed that in similarity with the ovary (Bockaert et al. 1976; Ezra & Salomon 1982a) desensitization of Leydig tumour cell plasma membrane adenylate cyclase may involve a GTP-mediated phosphorylation step.     

Seasonal changes in testicular structure and function in the blue fox (Alopex lagopus), as quantified by morphometric analysis and measurement of adenylate cyclase activity

The volume of the blue fox testis showed 5-fold changes during the year, associated with considerable changes in cellular composition. The seminiferous epithelium was maximally regressed in August, when 94% of tubules contained only spermatogonia. By late October, approximately 6 months before the mating season, 40% of tubules contained primary spermatocytes. From the middle of January until the end of April all tubules contained spermatids or more advanced haploid cells. Tubular diameter increased by 73% during testicular re-development, and epithelial height increased 3-fold. Regression to the basal state occurred during May to July. The volume densities of the seminiferous epithelium and of interstitial tissue remained approximately constant throughout the year. Soluble Mn2+-dependent adenylate cyclase activity showed seasonal variations that paralleled those of the haploid germ cell population and testicular volume, whereas somatic cell adenylate cyclase activity was relatively constant.     

Adenylate cyclase activity of mouse sperm during capacitation in vitro: effect of calcium and a GTP analogue

Earlier studies have shown increased adenylate cyclase (AC) activity in epididymal mouse sperm incubated under capacitating conditions in vitro. The present study investigated the effect on AC activity of excluding calcium and/or glucose from the sperm incubation medium, which would modulate expression of fertilizing potential. AC activity was higher in sperm incubated for 120 than 30 min, and was higher in sperm incubated in calcium-containing than calcium-free media for all except acrosome-reacted populations. Calcium added at the time of assay stimulated AC activity, the degree of this response being independent of the functional state of the sperm population. The guanine nucleotide analogue Gpp(NH)p slightly enhanced AC activity, but did not alter the stimulatory effect of calcium. Since calcium can increase AC activity, possibly by interaction with a divalent cation allosteric site on the catalytic subunit of the enzyme, a rise in intracellular calcium levels during capacitation may mediate the increased activity of AC, allowing expression of cAMP dependent events which are a prerequisite for fertilization.     

Elevation of alkaline phosphatase activity induced by parathyroid hormone in osteoblast-like cells from the spinal hyperostotic mouse TWY (twy/twy)

We have examined the alkaline phosphatase (AP) activity of primary calvaria-derived osteoblast-like cells from the twy (tip-toe walking Yoshimura) and normal ICR control mouse. The twy mouse displays elevated osseous formation particularly in the spine, and the pathophysiological features resemble that of human ankylosing spinal hyperostosis. In the proliferative stage of cultured bone cells, parathyroid hormone (PTH) stimulation induced the elevation of AP activity of both twy and ICR mouse-derived cells. When they reached confluence, the AP activity of ICR mouse-derived cells ceased to increase with PTH stimulation. The twy mouse-derived cells, however, continued to respond to PTH, with the enzyme activity increasing even in the confluent, stationary stage. PTH stimulation also increased the intracellular cAMP content of twy mouse-derived cells but it did not influence that of ICR mouse-derived cells in the stationary stage. Moreover, stimulation with dibutyryl cAMP, but not with phorbol myristate acetate, increased the AP activity of both twy and ICR-derived bone cells irrespective of culture conditions, either in the proliferative or in the confluent stage. These data suggest that the protein kinase A-mediated pathway plays a pivotal role in bone cells with PTH stimulation, and that the uninhibited AP activity observed in twy mouse-derived bone cells might be due to some deviating process between the PTH ligand/receptor interaction and cAMP generation.     

芦荟多糖对体外培养人表皮细胞增殖的影响

目的观察芦荟多糖(AP)对体外培养人表皮细胞增殖的影响。方法体外培养人表皮细胞,依据所用DK-SFM培养液中AP含量的不同,将细胞随机分为25、50、100、200和400mg/LAP组,对照组细胞仅加入等体积的DK-SFM培养液。通过倒置相差显微镜和透射电镜观察细胞形态和超微结构,并计算细胞融合时间。应用噻唑蓝法、氚标记胸腺嘧啶脱氧核苷(3H-TdR)掺入法、细胞计数法和流式细胞技术分别观察细胞存活率、3H-TdR掺入量、生长曲线分布情况及细胞周期,用以反映细胞增殖状况;通过检测乳酸脱氢酶(LDH)漏出率反映细胞损伤程度。结果倒置相差显微镜观察到,各组表皮细胞形态基本相似;透射电镜下观察到,100、400mg/L AP组细胞增殖活跃,核内以常染色质为主,而对照组和25mg/L AP组细胞则以异染色质为主。50~400mg/L AP组细胞融合时间分别为(154±12)、(141±20)、(130±19)、(124±13)h,明显早于对照组(182±8)h(P<0·01).从生长曲线上可见,100~400mg/LAP组细胞增殖达峰值的时间较其他组提前1~2d;25~400mg/LAP组细胞存活率、3H-TdR掺入量均高于对照组。与对照组比较,25~400mg/L AP组G0/G1期细胞所占百分比明显减少,G2/M期和S期细胞则明显增加(P<0.01).200、400mg/L AP组细胞LDH漏出率低于对照组及25、50mg/L AP组(P<0.01).结论高剂量AP对表皮细胞有保护效能,它通过诱导表皮细胞从G0/G1期进入G2/M期和S期促进细胞增殖。     

Regulation of hormone-induced cyclic AMP response to parathyroid hormone and prostaglandin E2 in cells cultured from human giant cell tumors of bone

Summary Cells dispersed from human giant cell tumors of bone and grown in monolayer culture increase intracellular cyclic AMP (cAMP) when incubated with parathyroid hormone (PTH) or prostaglandin E₂ (PGE₂). When cells are continuously exposed to PTH, cAMP levels increase acutely but then decrease rapidly to pretreatment values despite continued presence of hormone or addition of new hormone. Preincubation of cells with PTH for periods as short as 10 min results in a decrease in the capacity of cells to increase cAMP content when re-exposed to maximal stimulatory concentrations of PTH. The decrease in the magnitude of the PTH-induced cAMP response observed in cells pretreated with this hormone is dependent on the concentration of PTH present during the pre-incubation. The loss of cAMP response in cells pre-treated with either PGE₂ or PTH is hormone specific in that cells made refractory by pretreatment with one hormone still increase cAMP content when exposed to the other. Although the cells are not releasing measurable amounts of prostaglandins into the medium, pretreatment with indomethacin results in an increase in the magnitude of the cAMP response to PGE₂. The PTH-induced cAMP response is not affected by indomethacin pre-treatment. The loss of PTH responsiveness produced by hormone preincubation is consistent with the phenomenon of “down-regulation” observed with ligand-receptor interactions in a variety of tissues.     

黄芪甲苷对人皮肤成纤维细胞增殖和凋亡的影响

目的探讨黄芪甲苷对体外培养的有皱纹、无皱纹中老年人皮肤成纤维细胞生物学特性的影响.为黄芪甲苷用于延缓皮肤衰老和皱纹形成提供细胞水平的客观依据,为进一步探讨皮肤衰老机制和开发抗衰老护肤品提供新思路.方法体外培养中年人眼角皱纹与眼角无皱纹皮肤深处成纤维细胞;老年人面颊皮肤成纤维细胞.加入不同浓度的黄芪甲苷,干预24、72、120 h后,检测黄芪甲苷对这3例皮肤成纤维细胞系增殖活性、Ⅰ型胶原蛋白、Ⅲ型胶原蛋白、弹性蛋白合成和凋亡率的影响.结果黄芪甲苷在较低浓度（5～20μg/ml）时可促进皱纹和无皱纹皮肤成纤维细胞增殖,促进皱纹、无皱纹和老年皮肤成纤维细胞Ⅰ型胶原蛋白合成,降低皱纹和无皱纹皮肤成纤维细胞凋亡率（P＜0.01）.结论黄芪甲苷在较低浓度时具有改善皱纹和非皱纹皮肤成纤维细胞生物学特性的作用.