Calcium/calmodulin regulation of the proliferation of human epidermal keratinocytes, dermal fibroblasts and mouse B16 melanoma cells in culture

We have investigated the relationship between extracellular calcium, intracellular calmodulin and proliferation in normal keratinocytes. Keratinocyte proliferation and its sensitivity to calmodulin antagonists was compared with that of normal human dermal fibroblasts and neoplastic mouse B16 melanoma cells. Keratinocytes were similar to fibroblasts in showing reduced proliferation in low (0.15 mM) calcium medium and unlike B16 cells which continued to proliferate until calcium was reduced to submicromolar levels. Intracellular calmodulin was significantly higher in rapidly dividing keratinocytes in normal (1.15 mM) calcium medium than in slower dividing cells in low (0.15 mM) calcium. Fibroblasts and B16 cells maintained similar calmodulin levels in both low and normal calcium media. Calmodulin antagonists inhibited proliferation of all three cell types equally. Thus, keratinocyte calmodulin seems related to the proliferative state of the cell (unlike fibroblast calmodulin) and calmodulin antagonists may be of use in controlling the hyperproliferation of the psoriatic epidermis.     

Tonic Inhibition of TRPV3 by Mg(2+) in Mouse Epidermal Keratinocytes

The transient receptor potential vanilloid 3 channel (TRPV3) is abundantly expressed in epidermal keratinocytes and has important roles in sensory biology and skin health. Mg(2+) deficiency causes skin disorders under certain pathological conditions such as type 2 diabetes mellitus. In this study, we investigated the effect of Mg(2+) on TRPV3 in primary epidermal keratinocytes. Extracellular Mg(2+) ([Mg(2+)](o)) inhibited TRPV3-mediated membrane current and calcium influx. TRPV3 activation induced a calcium signaling pathway culminating in activation of the cAMP response element binding. TRPV3 inhibition by [Mg(2+)](o), the TRPV3 blocker ruthenium red, or TRPV3 siRNA suppressed this response. In TRPV3-expressing Chinese hamster ovary cells, both extracellular and intracellular Mg(2+) inhibited TRPV3 single-channel conductance, but not open probability. Neutralization of an aspartic acid residue (D641) in the extracellular pore loop or two acidic residues (E679, E682) in the inner pore region significantly attenuated the inhibitory effect of extracellular or intracellular Mg(2+) on TRPV3-mediated signaling, respectively. Our findings suggest that epidermal TRPV3 is tonically inhibited by both extracellular and intracellular Mg(2+), which act on both sides of the channel pore loop. Mg(2+) deficiency may promote the function of TRPV3 and contribute to the pathogenesis of skin diseases.     

Increased calmodulin levels in psoriasis and low Ca++ regulated mouse epidermal keratinocyte cultures

Calcium has been shown to regulate the proliferation of epidermal keratinocytes in vitro. We became interested in the role of the calcium binding protein, calmodulin, in hyperproliferative, low calcium regulated keratinocytes in vitro and in the in vivo hyperproliferative state, psoriasis. Calmodulin levels were measured by radioimmune assay in neonatal mouse keratinocytes grown in 0.02 mM calcium (hyperproliferative) and 1.2 mM calcium (normal) media, and in cells that had been grown in low calcium medium and then switched to normal calcium. On a whole culture basis the normal cells had more calmodulin than the low calcium cells. However, when low calcium monolayers were compared to the normal basal monolayer, the low calcium hyperproliferative cells had more calmodulin. Cells that were switched from 0.02 mM calcium to 1.2 mM calcium showed increasing calmodulin levels over time. Psoriatic plaques contained 2-3 times more calmodulin than the skin of normal controls when examined on a per micrograms of DNA, per micrograms of protein, and per gram of wet weight basis. Adjacent uninvolved psoriatic skin also had significantly elevated calmodulin levels in all data bases except per microgram of protein/cm2. These data suggest that increased calmodulin levels are associated with epidermal hyperproliferation and/or with the state of differentiation.     

Papaverine and Ro 20-1724 inhibit cyclic nucleotide phosphodiesterase activity and increase cyclic AMP levels in psoriatic epidermis in vitro.

The comparative inhibitory potency of papaverine and Ro 20-1724 (4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone) on cyclic AMP-phosphodiesterase (cAMP-PDE) and cyclic GMP-phosphodiesterase (cGMP-PDE) activities and their effect on the levels of cAMP and cGMP were examined in psoriatic epidermis. At concentrations of 5 X 10(-4) M, papaverine inhibited the hydrolysis of both cAMP and cGMP by either the low or high Km psoriatic epidermal PDE nearly 100% (p less than .0001) while Ro 20-1724 selectively inhibited the hydrolysis of cAMP 94% (p less than .0001) but had no significant effect on cGMP hydrolysis. When keratomed psoriatic epidermal slices were incubated in 5 X 10(-4) M papaverine or Ro 20-1724 the tissue levels of cAMP were increased 343% or 1395% respectively (p less than .001) with no concomitant change in the levels of cGMP. Selective inhibition of cAMP hydrolysis by Ro 20-1724 and its greater effectiveness in elevating cAMP levels in slices of psoriatic epidermis is one explanation for its clinical superiority in treating psoriatic lesions.     

Epidermolysis bullosa dystrophica recessive fibroblasts produce increased concentrations of cAMP within a collagen matrix

Human dermal fibroblasts grown in tissue culture can be suspended and cultured in collagen lattices. These fibroblast-populated collagen lattices (FPCL) undergo a reduction in size by the process of lattice contraction. Fibroblasts from patients with epidermolysis bullosa dystrophica recessive, EBdr, produce excessive quantities of cAMP. These high concentrations of cAMP may be related to the inability of the EBdr fibroblast to elongate and spread out when incorporated into the collagen matrix. Fibroblasts with these morphologic characteristics are not effective in contracting collagen lattices. EBdr fibroblasts in FPCL have intracellular concentrations of cAMP 8 times greater than those of normal fibroblasts in FPCL. They also have a dendritic morphology. The addition of cholera toxin or dibutyryl cAMP to normal human fibroblasts will cause elevated levels of intracellular cAMP and will inhibit the elongation and spreading of cells and lattice contraction. The cytoskeletal morphology of EBdr fibroblasts differs from that of normal human fibroblasts in FPCL. The use of rhodamine phalloidin, a specific fluorescent stain for F-actin filaments, reveals that EBdr fibroblasts show a pattern of actin distribution shared by normal fibroblasts cultured in the presence of dibutyryl cAMP or cholera toxin. It is proposed that the contractile forces responsible for lattice contraction are identical to those forces responsible for the spreading and elongation of cells. EBdr fibroblasts fail to spread and elongate within a collagen matrix and are therefore not effective in lattice contraction.     

Effect of protein kinase C on transmembrane calcium fluxes in HaCaT keratinocytes

Capacitive calcium influx is associated with the release of calcium from internal stores and participates in intracellular calcium homeostasis. In keratinocytes, its activation is linked to the stimulation of the phospho-inositide (PI) pathway and seems to be altered in psoriasis. An overnight treatment of isolated HaCaT keratinocytes with phorbol 12-myristate 13-acetate (PMA) selectively downregulated the classical, calcium-dependent protein kinase C (PKC) isoenzyme PKC alpha in preconfluent cells. This was parallelled by an increased capacitative calcium influx with no effects on the PI pathway. These observations were strengthened in measurements using cyclopiazonic acid which revealed a 47% increase in PMA pretreated as compared with control cells in the calcium influx rate through store-operated calcium channels (SOC-s) following the emptying of the intracellular calcium stores. In confluent as compared with preconfluent cultures PKC epsilon was markedly increased, while other isoenzymes were not affected. In parallel, the kinetics of capacitative calcium influx were altered, showing clear inactivation. PMA pretreatment in these cells had little effect on PKC alpha but downregulated both PKC beta and PKC epsilon, and did not increase the influx through SOC-s. These observations support the differential regulation of SOC-s by PKC and suggest the involvement of several PKC isoenzymes in human keratinocytes.     

The role of iron in an acute model of skin inflammation induced by reactive oxygen species (ROS)

The effect of iron was studied in rats in a ROS-initiated model of acute skin inflammation. Iron dextran was administered i.v. 24 h before the induction of the inflammatory response by intradermal injection of glucose oxidase attached to polyethylene glycol (GOD-PEG). Iron exacerbated the response at 24 and 48 h (P greater than 0.001). Histologically, a similar picture was seen to that without iron except for an increase in tissue oedema and matrix destruction including the skin glands. Associated with iron loading was an increase in Perls stainable iron in the skin (P greater than 0.025) and liver (P greater than 0.001). However, skin inflammation without iron loading also increased skin iron levels (P greater than 0.025). Total serum iron was decreased in iron-loaded and GOD-PEG animals (P greater than 0.01) and the unbound iron binding capacity (UIBC) increased (P greater than 0.01).     

Local absorption of zinc from wounds treated with different concentrations of zinc sulphate.

In the present study it was shown in rats that zinc is absorbed from excisional wounds treated with zinc sulphate. Systemic toxic effects were observed in the group treated with 20% zinc sulphate. Local toxic effects were seen in wounds treated with 0.2%, 2% and 20% zinc sulphate. An inhibitory effect of zinc on the migration of granulocytes was suggested on the basis of microscopic observation. In the operated groups which were not treated with zinc and the group treated with 0.02% zinc sulphate a decline was observed in the concentration of zinc in serum. The zinc concentration in serum increased in proportion to the zinc sulphate concentration (0.2%, 2% and 20%) applied to the wounds, while the copper concentration decreased in the groups treated with 2% and 20% zinc sulphate. In all operated groups an increase in zinc and copper concentrations was observed in liver. This was most pronounced in groups treated with higher concentrations of zinc sulphate (0.2%, 2% and 20%). The groups treated with higher concentrations of zinc sulphate also had higher pancreas zinc concentrations than the remaining groups.     

白介素-10对小鼠光变态反应性接触性皮炎的影响

目的：探讨白介素(IL)-10在光变态反应性接触性皮炎(photoallergic contact dermatitis，PACD)发病中的作用。方法：选用BALB／C纯系雌性小鼠建立氯丙嗪(chlorpromazine，CPZ)所致的PACD模型，采用腹腔或皮内注射重组小鼠IL-10的方法，于激发48h后检测小鼠耳肿度，局部皮损活检行苏木精-伊红染色，计数单一核细胞浸润数。结果：小鼠皮内和腹腔注射重组小鼠IL-10后，耳肿度值、单一核细胞浸润数均较阳性对照组(单纯的PACD模型)低。结论：小鼠体内IL-10对CPZ诱导的PACD的光诱导和光激发阶段均起抑制作用。     

Effect of topical application and intraperitoneal injection of oregonin on atopic dermatitis in NC/Nga mice

Please cite this paper as: Effect of topical application and intraperitoneal injection of oregonin on atopic dermatitis in NC/Nga mice. Experimental Dermatology 2010; 19 : e37–e43. Abstract: The diarylheptanoid, oregonin (ORE), which was isolated from the bark of Alnus japonica Steudel that grows natively in Korea, has been known to exert antioxidative, anti‐inflammatory, anti‐cancer and immune response inhibitory effects. The antioxidative effect of ORE was observed on the superoxide and 1,1‐diphenyl‐2‐picrylhydrazyl radical, as well as on the expression of inducible nitric oxide synthase and cyclooxygenase‐2 in lipopolysaccharide‐treated RAW264.7 macrophages. The statistically significant inhibitory action of ORE against production of cytokines induced by bacterial products or by interleukin (IL)‐1β, free radicals and nitrogen species, and a corresponding increase in cellular calcium concentration because of ORE were confirmed in bone marrow and spleen dendritic cells that are known to play important functions in the development and advancement of atopic dermatitis (AD). It was thus expected that ORE would exert a beneficial effect in the treatment of AD. A study on the pharmaceutical benefits of ORE against AD has not yet been conducted in vivo. We therefore used an in vivo AD animal model, namely the NC/Nga mice, and by applying ORE onto the animals through skin application as well as intraperitoneal injection, we attempted to evaluate the benefits of ORE in this system. Evaluation of ORE was conducted by following the SCORE method to score the effect, as well as by measuring the Th2 cytokines IL‐4, IL‐5 and IL‐13 levels from serum and lymphocytes, and IgE and eosinophil levels from serum. Additionally, the expression of mRNA and protein levels was estimated using real‐time polymerase chain reaction and Western blotting analysis. The following categories of clinical evaluation, Th2 cytokines IL‐4, IL‐5 and IL‐13 values, serum IgE levels, serum eosinophil levels, and mRNA and protein expression levels of iNOS and COX‐2, were evaluated from topical application and intraperitoneal injection groups of ORE. The effects of ORE on AD in NC/Nga mice were confirmed as being similar to the positive control group, while a significant difference with the negative control group was observed. The results presented in this report suggest that ORE might be beneficial in the treatment of AD.     

Zinc and genital infections.

The zinc status of 19 patients with chronic or recurrent genital infections and 18 patients with non-recurrent genital infections was assessed by measuring plasma and leucocyte zinc concentrations. Neither group of patients had plasma or leucocyte zinc concentrations that differed significantly from those of matched healthy controls. Each of six patients with chronic candidiasis had anergy to candidal antigen, as shown by delayed cutaneous hypersensitivity to intradermal injection of the antigen, but their zinc status was normal. This study provided no evidence of zinc deficiency in this small number of patients with acute non-recurrent or chronic recurrent genital infections.     

CD44, but not l-selectin, is critically involved in leucocyte migration into the skin in a murine model of allergic dermatitis

CD44 and l-selectin (CD62L) are major adhesion receptors that mediate leucocyte recruitment at inflammatory sites and lymph nodes, by supporting cell rolling under blood flow. Both CD44 and CD62L have been implicated in inflammatory skin disorders, but their specific involvement in an immediate-type allergic reaction remains uncertain. We used mice deficient in CD44 or CED62L or both in order to determine whether one or both of these molecules were required for leucocyte extravasation in an atopic dermatitis-like allergic response. Wild-type (WT) mice and mice deficient in CD44, CD62L or both were immunized with ovalbumin (OVA). Inflammatory reaction in the ear was elicited once by means of intradermal injection of OVA. Effective sensitization of CD62L knockout (KO) mice required intraperitoneal antigen injection; however, OVA-specific T helper 2 (Th2)-type immune responses and IgE production in mice lacking CD44, CD62L or both were comparable to those in WT mice following intraperitoneal immunization. We employed intravital videomicroscopy to monitor the recruitment of fluorescence-labelled leucocytes to the ear tissue following challenge with OVA. The number of adherent leucocytes was significantly reduced in CD44 KO and CD44/CD62L double KO mice, indicating that CD44 was involved in firm adhesion, the committed step of leucocyte extravasation. Histology of the OVA-challenged ears showed a diminished leucocyte infiltration in the ears of CD44 KO and double KO mice. The results of our study demonstrate that CD44, but not CD62L, is required for leucocyte extravasation during a Th2-type inflammatory response.     

N-acetyl cysteine promotes angiogenesis and clearance of free oxygen radicals,thus improving wound healing in an alloxan-induced diabetic mouse model of incisional wound

Background. This study investigated whether N-acetyl cysteine induces any favourable effects on cutaneous incisional wound healing in diabetic and nondiabetic mice. The wounds were assessed using detection of vascular endothelial growth factor (VEGF) and inducible nitric oxide synthase (iNOS) expression, and wound-breaking strength (WBS) measurements. Methods. In total, 48 BALB/c mice were used. These were divided into four groups, each consisting of 12 mice. Incisional wounds were made on the back of each mouse. Two of the groups consisted of healthy animals and the other two groups consisted of mice with alloxan-induced diabetes. One group of healthy mice and one group of diabetic mice received intraperitoneal N-acetyl cysteine (NAC) 150 mg/kg for 5 consecutive days, while the other two groups were untreated. On the fifth day all animals were killed, and the WBS, oxidative stress parameters, histopathological and immunohisotchemical results were assessed. Results. Both diabetic and nondiabetic mice receiving NAC had lower levels of oxidative stress markers and higher WBS measurements than untreated counterparts. Conclusions. A mouse model of incisional wound treated with NAC resulted in lower levels of tissue oxidative stress, higher levels of tissue glutathione, and downregulation of iNOS expression coupled with upregulation of VEGF expression, producing an overall favourable clinical outcome of higher WBS and a shorter wound-healing period both in diabetic and nondiabetic mice. Both antioxidant and anti-inflammatory properties of NAC may be involved in this improved healing process for incisional wounds.     

Association of cyclic adenosine monophosphate with permeability barrier homeostasis of murine skin

Activation of Gs protein increases the intracellular cyclic adenosine monophosphate (cAMP) level, and the Gs protein-linked receptor has been implicated in the skin barrier homeostasis. In this study, we investigated the role of cAMP in epidermal barrier function. The barrier was disrupted by tape stripping or treatment with acetone. Immediately after barrier disruption, reagents affecting the cAMP level were topically applied. Topical application of forskolin, which activates cAMP synthesis delayed barrier recovery, whereas application of the antagonist of cAMP, cAMP-Rp, accelerated barrier recovery. Moreover, application of 9-cyclopentyladenine, an inhibitor of cAMP synthesis also accelerated barrier recovery. Tape stripping was found to increase the cAMP in the epidermis. Light and electron microscopic observations showed the delay of lamellar body secretion by forskolin and acceleration of the lamellar body secretion by cAMP-Rp. Application of an inhibitor of protein kinase A did not affect the barrier recovery rate. The delay of barrier recovery induced by forskolin was blocked by the voltage-gated calcium channel blockers, nifedipine and verapamil. In cultured keratinocytes, forskolin increased the intracellular calcium concentration and both nifedipine and verapamil blocked the increase. These results suggest that intracellular cAMP in the epidermis is involved in skin barrier homeostasis.     

Topical treatment of recurrent herpes simplex and post-herpetic erythema multiforme with low concentrations of zinc sulphate solution

The preventive effect of low concentrations of zinc sulphate solution in recurrent herpes simplex of the skin and oral mucous membrane is reported. Treatment with zinc sulphate solution of the skin at the site of the herpetic infection also prevents relapse of post-herpetic erythema multiforme. For the skin, 0.025-0.05%, and for the oral mucous membrane, 0.01-0.025% zinc sulphate solution was used.     

Cyclic AMP and psoriasis once more.

Imbalanced cyclic nucleotides are implicated as an underlying pathogenetic mechanism in psoriasis, but conflicting data on the levels of cAMP in psoriatic tissue have been obtained by different laboratories. Using heat separation and a competitive protein binding cAMP assay, a noticeably decreased epidermal level of cAMP was detected in newly formed guttate lesions in 10 patients with psoriasis (involved: 4.32 +/- 1.06 pmol/mg dry weight mean +/- S.E.M.). uninvolved: 7.97 +/- 1.63 pmol/mg dry weight mean +/- S.E.M.). In proliferative rat skin, bidirectional alterations in cAMP levels are known to occur. On the basis of present observation, we assume that the conflicting data hitherto obtained in psoriatic tissue similarly may merely reflect the various levels of cAMP at different developmental stages.     

ATP-induced cell contraction with epidermolysis bullosa dystrophica recessive and normal dermal fibroblasts

Human dermal fibroblasts cultured on glass coverslips and permeabilized by glycerol can be induced to undergo cell shrinkage by the addition of ATP in buffer containing calcium and magnesium. They reduce in size by 72% in 10 min. ATP-induced cell contraction is linked to an aggregation of cytoplasmic filaments as demonstrated by rhodamine-phalloidin F-actin staining. Before the addition of ATP, glycerinated cells have parallel arrangements of staining cytoplasmic filaments. Afterward, dermal fibroblasts from patients with epidermolysis bullosa dystrophica recessive (EBdr) show only a 10-20% reduction in cell size, and little F-actin aggregation staining can be demonstrated. Epidermolysis bullosa dystrophica recessive fibroblasts have been reported to produce excess prostaglandin E2 (PGE2) and cAMP. The preincubation of normal dermal fibroblasts for 24-30 h with 10 micrograms/ml PGE2, 10 micrograms/ml cholera toxin, or 1 mM dibutyl cAMP will reduce ATP-induced cell contraction to less than 20%. Treated cells showed little disruption of cytoplasmic F-actin. Epidermolysis bullosa dystrophica recessive fibroblasts preincubated with the cyclooxygenase inhibitor indomethacin at 10 micrograms/ml restored cell contraction to 74%. These treated cells also show aggregation of F-actin filaments. The process of ATP-induced cell contraction can be altered by the intracellular concentrations of cAMP, the levels of which are elevated in the fibroblasts in EBdr patients. A mechanism for cAMP inhibition of cell contraction is discussed.     

二丁酰环磷腺苷钙联合阿维A治疗寻常型银屑病的疗效及对血cAMP和cGMP的影响

目的：观察二丁酰环磷腺苷钙联合阿维A治疗寻常型银屑病的疗效及对血cAMP和cGMP的含量影响。方法：将64例寻常型银屑病患者随机分为治疗组（32例）和对照组（32例）,治疗组予以二丁酰环磷腺苷钙60mg加入5%葡萄糖注射液250 mL中静滴,1次/天,阿维A胶囊10 mg口服,3次/天,对照组单用阿维A胶囊。并对两组治疗前后血浆中cAMP和cGMP的含量进行测定。结果：治疗组总有效率83.4%,对照组总有效率54.3%,两组综合疗效及治疗后PASI评分比较差异有统计学意义（P〈0.05）;治疗后两组cAMP、cGMP含量比较差异有统计学意义（P〈0.05）。结论：采用二丁酰环磷腺苷钙联合阿维A治疗寻常型银屑病效果满意,是一种可供临床选择的治疗方案。     

Psoriasis and altered calcium metabolism: downregulated capacitative calcium influx and defective calcium-mediated cell signaling in cultured psoriatic keratinocytes

Intracellular calcium plays an important part in the regulation of proliferation and differentiation of keratinocytes. Detached from their in vivo environment, cultured psoriatic keratinocytes were investigated by monitoring free intracellular calcium concentration, which was measured using fura-2/AM as a calcium-sensitive probe. The mean increase in intracellular calcium of psoriatic keratinocytes was significantly reduced compared with control keratinocytes when intracellular calcium stores were mobilized from endoplasmic reticulum with thapsigargin. This finding suggests defective capacitative calcium influx of psoriatic cells. Intracellular calcium stores were similar in psoriatic and control keratinocytes, when extracellular calcium was chelated with ethyleneglycol-bis(beta-aminoethyl ether)-N,N,N',N',-tetraacetic acid and intracellular calcium was depleted with thapsigargin. Mechanical wounding of keratinocyte monolayer resulted in a significantly reduced rise in intracellular calcium of psoriatic cells in low (< 0.1 mM) and high (1.8 mM) extracellular calcium suggesting defective intercellular coupling of psoriatic keratinocytes. Blocking of gap-junctions with heptanol in wounded keratinocytes did not affect the intracellular calcium response in psoriatic keratinocytes in contrast to healthy keratinocytes. Adding adenosine triphosphate to culture medium resulted in a more pronounced intracellular calcium increase than thapsigargin in psoriatic keratinocytes, suggesting that inositol triphosphate-mediated, P2-purinergic signaling was enhanced in these cells. Moreover, psoriatic keratinocytes maintained their defective responses up to at least fifth passage suggesting that psoriatic keratinocytes have an inborn error in calcium metabolism, rather than a localized defect in response to altered extracellular calcium gradient observed in vivo.     

尘螨变应原rDer p1诱导HaCaT细胞表达胸腺基质淋巴生成素的研究

【摘要】 目的 探讨尘螨变应原rDer p1在体外对角质形成细胞表达胸腺基质淋巴生成素（TSLP）的影响。 方法 体外培养人角质形成细胞株HaCaT细胞,予以0.1、1和10 mg/L尘螨变应原rDer p1与蛋白酶活化受体2（PAR2）特异性激动剂SLGIKV（500 μmol/L）和拮抗剂VKGILS（500 μmol/L）共培养,以无血清培养基为空白对照。用ELISA法和荧光定量PCR法检测各实验组和对照组TSLP蛋白及mRNA表达水平;通过激光共聚焦显微镜检测细胞内钙流变化分析rDer p1诱导HaCaT细胞产生TSLP和PAR2受体活化的关系。 结果 1 mg/L和10 mg/L rDer p1组培养12 h上清中TSLP水平分别为（155.5 ± 5.9） ng/L和（228.8 ± 28.7） ng/L,显著高于空白对照组（54.3 ± 13.9 ng/L,P < 0.01）,TSLP表达水平随rDer p1浓度增加而升高。SLGIKV组TSLP表达［（166.2 ± 8.8） ng/L］也显著高于空白对照组（P < 0.01）。10 mg/L rDer p1组和SLGIKV组TSLP mRNA相对表达量在8 h最高,分别为（3.28 ± 0.27）倍和（2.15 ± 0.26）倍,与4 h和24 h相比差异有统计学意义（P < 0.01）。SLGIKV可引起HaCaT细胞钙内流增加;10 mg/L rDer p1也可引起HaCaT细胞钙内流增加,经过PAR2受体特异性阻断剂VKGILS（500 μmol/L）处理后再给予rDer p1刺激,细胞内钙内流峰值明显降低。 结论 尘螨变应原rDer p1能够通过部分活化HaCaT细胞表面的PAR2受体诱导产生促炎细胞因子TSLP。