TRPA1 mediates bladder hyperalgesia in a mouse model of cystitis

Urinary bladder pain is a primary symptom associated with interstitial cystitis/painful bladder syndrome. We used systemic injections of cyclophosphamide (CYP), an alkylating antineoplastic agent, to induce cystitis and examine the roles of 2 channels previously demonstrated to be required for inflammatory visceral hyperalgesia: transient receptor potential vanilloid-1 (TRPV1) and ankyrin-1 (TRPA1). Injection of CYP (100 mg/kg, i.p.) every other day for 5 days was accompanied by bladder edema and urothelial ulceration, but without significant plasma extravasation or infiltration of neutrophils. Toluidine blue staining showed a significant increase in the number of degranulated bladder mast cells after CYP treatment. Despite this mild pathology, CYP-treated mice exhibited bladder hyperalgesia 1 day after the final injection that persisted 7 days later. Although many previous studies of visceral hyperalgesia have reported changes in dorsal root ganglion neuron TRPV1 expression and/or function, we found no change in bladder afferent TRPV1 expression or sensitivity on the basis of the percentage of bladder afferents responsive to capsaicin, including at submaximal concentrations. In contrast, the percentage of bladder afferents expressing functional TRPA1 protein (ie, those responsive to mustard oil) increased ∼2.5-fold 1 day after CYP treatment, and remained significantly elevated 7 days later. Moreover, bladder hyperalgesia was reversed by acute treatment with the TRPA1 antagonist HC-030031 (300 mg/kg, i.p.). Our results indicate that CYP-induced bladder hyperalgesia can be induced without robust inflammation or changes in primary afferent TRPV1. However, significant changes were observed in TRPA1 expression, and blockade of TRPA1 alleviated CYP-induced bladder hyperalgesia.     

Petasin and isopetasin reduce CGRP release from trigeminal afferents indicating an inhibitory effect on TRPA1 and TRPV1 receptor channels

BackgroundButterbur root extract with its active ingredients petasin and isopetasin has been used in the prophylactic treatment of migraine for years, while its sites of action are not completely clear. Calcitonin gene-related peptide (CGRP) is known as a biomarker and promoting factor of migraine. We set out to investigate the impact of petasins on the CGRP release from trigeminal afferents induced by activation of the calcium conducting transient receptor potential channels (TRPs) of the subtypes TRPA1 and TRPV1.MethodsWe used well-established in vitro preparations, the hemisected rodent skull and dissected trigeminal ganglia, to examine the CGRP release from rat and mouse cranial dura mater and trigeminal ganglion neurons, respectively, after pre-incubation with petasin and isopetasin. Mustard oil and capsaicin were used to stimulate TRPA1 and TRPV1 receptor channels. CGRP concentrations were measured with a CGRP enzyme immunoassay.ResultsPre-incubation with either petasin or isopetasin reduced mustard oil- and capsaicin-evoked CGRP release compared to vehicle in an approximately dose-dependent manner. These results were validated by additional experiments with mice expressing functionally deleted TRPA1 or TRPV1 receptor channels.ConclusionsEarlier findings of TRPA1 receptor channels being involved in the site of action of petasin and isopetasin are confirmed. Furthermore, we suggest an important inhibitory effect on TRPV1 receptor channels and assume a cooperative action between the two TRP receptors. These mechanisms may contribute to the migraine prophylactic effect of petasins.     

Supraspinal TRPV1 modulates the emotional expression of abdominal pain

The transient receptor potential vanilloid receptor type–1 (TRPV1) is critically involved in peripheral nociceptive processes of somatic and visceral pain. However, the role of the capsaicin receptor in the brain regarding visceral pain remains elusive. Here, we studied the contribution of TRPV1 to abdominal pain transmission at different nociceptive pathway levels using TRPV1 knock-out mice, resiniferatoxin-mediated deletion of TRPV1-positive primary sensory neurons, and intracerebral TRPV1 antagonism. We found that constitutive genetic TRPV1 deletion or peripheral TRPV1 deletion reduced acetic acid–evoked abdominal constrictions, without affecting referred abdominal hyperalgesia or allodynia in an acute pancreatitis model of visceral pain. Notably, intracerebral TRPV1 antagonism by SB 366791 significantly reduced chemical and inflammatory spontaneous abdominal nocifensive responses, as observed by reduced expressions of nociceptive facial grimacing, illustrating the affective component of pain. In addition to the established role of cerebral TRPV1 in anxiety, fear, or emotional stress, we demonstrate here for the first time that TRPV1 in the brain modulates visceral nociception by interfering with the affective component of abdominal pain.     

Isopentenyl pyrophosphate is a novel antinociceptive substance that inhibits TRPV3 and TRPA1 ion channels

Transient receptor potential ion channels (TRPs) expressed in the periphery sense and electrically transduce noxious stimuli to transmit the signals to the brain. Many natural and synthetic ligands for the sensory TRPs have been found, but little is known about endogenous inhibitors of these TRP channels. Recently, we reported that farnesyl pyrophosphate, an endogenous substance produced in the mevalonate pathway, is a specific activator for TRPV3. Here, we show that isopentenyl pyrophosphate (IPP), an upstream metabolite in the same pathway, is a dual inhibitor for TRPA1 and TRPV3. By using Ca²⁺ imaging and voltage clamp experiments with human embryo kidney cell heterologous expression system, cultured sensory neurons, and epidermal keratinocytes, we demonstrate that micromolar IPP suppressed responses to specific agonists of TRPA1 and TRPV3. Consistently, peripheral IPP administration attenuated TRPA1 and TRPV3 agonist-specific acute pain behaviors. Furthermore, local IPP pretreatment significantly reversed mechanical and thermal hypersensitivity of inflamed animals. Taken together, the present study suggests that IPP is a novel endogenous TRPA1 and TRPV3 inhibitor that causes local antinociception. Our results may provide useful chemical information to elucidate TRP physiology in peripheral pain sensation.     

Parthenolide inhibits nociception and neurogenic vasodilatation in the trigeminovascular system by targeting the TRPA1 channel

Although feverfew has been used for centuries to treat pain and headaches and is recommended for migraine treatment, the mechanism for its protective action remains unknown. Migraine is triggered by calcitonin gene-related peptide (CGRP) release from trigeminal neurons. Peptidergic sensory neurons express a series of transient receptor potential (TRP) channels, including the ankyrin 1 (TRPA1) channel. Recent findings have identified agents either inhaled from the environment or produced endogenously that are known to trigger migraine or cluster headache attacks, such as TRPA1 simulants. A major constituent of feverfew, parthenolide, may interact with TRPA1 nucleophilic sites, suggesting that feverfew’s antimigraine effect derives from its ability to target TRPA1. We found that parthenolide stimulates recombinant (transfected cells) or natively expressed (rat/mouse trigeminal neurons) TRPA1, where it, however, behaves as a partial agonist. Furthermore, in rodents, after initial stimulation, parthenolide desensitizes the TRPA1 channel and renders peptidergic TRPA1-expressing nerve terminals unresponsive to any stimulus. This effect of parthenolide abrogates nociceptive responses evoked by stimulation of peripheral trigeminal endings. TRPA1 targeting and neuronal desensitization by parthenolide inhibits CGRP release from trigeminal neurons and CGRP-mediated meningeal vasodilatation, evoked by either TRPA1 agonists or other unspecific stimuli. TRPA1 partial agonism, together with desensitization and nociceptor defunctionalization, ultimately resulting in inhibition of CGRP release within the trigeminovascular system, may contribute to the antimigraine effect of parthenolide.     

TRPM8 is the principal mediator of menthol-induced analgesia of acute and inflammatory pain

Menthol, the cooling natural product of peppermint, is widely used in medicinal preparations for the relief of acute and inflammatory pain in sports injuries, arthritis, and other painful conditions. Menthol induces the sensation of cooling by activating TRPM8, an ion channel in cold-sensitive peripheral sensory neurons. Recent studies identified additional targets of menthol, including the irritant receptor, TRPA1, voltage-gated ion channels and neurotransmitter receptors. It remains unclear which of these targets contribute to menthol-induced analgesia, or to the irritating side effects associated with menthol therapy. Here, we use genetic and pharmacological approaches in mice to probe the role of TRPM8 in analgesia induced by L-menthol, the predominant analgesic menthol isomer in medicinal preparations. L-menthol effectively diminished pain behavior elicited by chemical stimuli (capsaicin, acrolein, acetic acid), noxious heat, and inflammation (complete Freund’s adjuvant). Genetic deletion of TRPM8 completely abolished analgesia by L-menthol in all these models, although other analgesics (acetaminophen) remained effective. Loss of L-menthol–induced analgesia was recapitulated in mice treated with a selective TRPM8 inhibitor, AMG2850. Selective activation of TRPM8 with WS-12, a menthol derivative that we characterized as a specific TRPM8 agonist in cultured sensory neurons and in vivo, also induced TRPM8-dependent analgesia of acute and inflammatory pain. L-menthol– and WS-12–induced analgesia was blocked by naloxone, suggesting activation of endogenous opioid-dependent analgesic pathways. Our data show that TRPM8 is the principal mediator of menthol-induced analgesia of acute and inflammatory pain. In contrast to menthol, selective TRPM8 agonists may produce analgesia more effectively, with diminished side effects.     

Homologous and heterologous desensitization of capsaicin and mustard oil responses utilize different cellular pathways in nociceptors

The transient receptor potential channel subtypes V1 (TRPV1) and A1 (TRPA1) play a critical role in the development of hyperalgesia in inflammatory pain models. Although several studies in animals and humans have demonstrated that capsaicin (CAP), a TRPV1-specific agonist, and mustard oil (MO), a TRPA1 agonist, evoke responses that undergo functional cross-desensitization in various models, the mechanisms mediating this phenomenon are largely unknown. In the present study, we evaluated the mechanisms underlying homologous and heterologous desensitization between CAP and MO responses in peripheral nociceptors using an in vitro neuropeptide release assay from acutely isolated rat hindpaw skin preparation and in vivo behavioral assessments. The pretreatment with CAP or MO significantly inhibited (50-60%) both CAP- and MO-evoked CGRP release indicating homologous and heterologous desensitization using this assay. Further studies evaluating the requirement of calcium in these phenomena revealed that homologous desensitization of CAP responses was calcium-dependent while homologous desensitization of MO responses was calcium-independent. Moreover, heterologous desensitization of both CAP and MO responses was calcium-dependent. Further studies evaluating the role of calcineurin demonstrated that heterologous desensitization of CAP responses was calcineurin-dependent while heterologous desensitization of MO responses was calcineurin-independent. Homologous and heterologous desensitization of CAP and MO was also demonstrated using in vivo behavioral nocifensive assays. Taken together, these results indicate that TRPV1 and TRPA1 could be involved in a functional interaction that is regulated via different cellular pathways. The heterologous desensitization of these receptors and corresponding inhibition of nociceptor activity might have potential application as a therapeutic target for developing novel analgesics.     

The involvement of the transient receptor potential A1 (TRPA1) in the maintenance of mechanical and cold hyperalgesia in persistent inflammation

This study investigated the role of TRPA1 in the development and maintenance of mechanical and cold hyperalgesia in persistent inflammation induced by Complete Freund’s Adjuvant (CFA) in mice. The intraplantar (i.pl.) injection of CFA induced a long lasting (28 days) hyperalgesia for both mechanical and thermal (cold) stimuli. The intraperitoneal (i.p., 30–300 mg/kg), intraplantar (i.pl., 100 μg/site) or intrathecal (i.t., 10 μg/site) injection of the TRPA1 selective antagonist HC-030031 significantly reduced the mechanical hyperalgesia evaluated by the von Frey hair test. The effect of HC-030031 was evidenced on the day after CFA injection and was kept throughout the test. However, the intracerebroventricular (i.c.v., 10 μg/site) injection of HC-030031 did not interfere with CFA-induced hyperalgesia. Treatment with HC-030031 (300 mg/kg, i.p.) completely inhibited the noxious cold hyperalgesia induced by tetrafluoroethane in mice that received CFA. The pre-treatment with the TRPA1 oligonucleotide antisense (AS-ODN, i.t.) consistently prevented both mechanical and cold hyperalgesia. Interestingly, both TRPA1 protein expression and mRNA were over-expressed in spinal cord and dorsal root ganglia (DRG) of mice treated with CFA, an effect that was fully prevented by the pre-treatment with the TRPA1 antagonist HC-030031. Collectively, the present results showed that TRPA1 present at either peripheral or spinal sites play a relevant role in the development and maintenance of both mechanical and cold hyperalgesia during CFA-induced inflammation. Thus, TRPA1 selective antagonists represent promising candidates to treat hyperalgesia in persistent inflammatory states.     

Oxaliplatin elicits mechanical and cold allodynia in rodents via TRPA1 receptor stimulation

Platinum-based anticancer drugs cause neurotoxicity. In particular, oxaliplatin produces early-developing, painful, and cold-exacerbated paresthesias. However, the mechanism underlying these bothersome and dose-limiting adverse effects is unknown. We hypothesized that the transient receptor potential ankyrin 1 (TRPA1), a cation channel activated by oxidative stress and cold temperature, contributes to mechanical and cold hypersensitivity caused by oxaliplatin and cisplatin. Oxaliplatin and cisplatin evoked glutathione-sensitive relaxation, mediated by TRPA1 stimulation and the release of calcitonin gene-related peptide from sensory nerve terminals in isolated guinea pig pulmonary arteries. No calcium response was observed in cultured mouse dorsal root ganglion neurons or in naïve Chinese hamster ovary (CHO) cells exposed to oxaliplatin or cisplatin. However, oxaliplatin, and with lower potency, cisplatin, evoked a glutathione-sensitive calcium response in CHO cells expressing mouse TRPA1. One single administration of oxaliplatin produced mechanical and cold hyperalgesia in rats, an effect selectively abated by the TRPA1 antagonist HC-030031. Oxaliplatin administration caused mechanical and cold allodynia in mice. Both responses were absent in TRPA1-deficient mice. Administration of cisplatin evoked mechanical allodynia, an effect that was reduced in TRPA1-deficient mice. TRPA1 is therefore required for oxaliplatin-evoked mechanical and cold hypersensitivity, and contributes to cisplatin-evoked mechanical allodynia. Channel activation is most likely caused by glutathione-sensitive molecules, including reactive oxygen species and their byproducts, which are generated after tissue exposure to platinum-based drugs from cells surrounding nociceptive nerve terminals.     

Changes in vanilloid receptor 1 (TRPV1) expression following lingual nerve injury.

We have investigated a possible role for vanilloid receptor 1 (TRPV1), a transducer of noxious stimuli, in the development of neuropathic pain following injury to a peripheral branch of the trigeminal nerve. In nine adult ferrets the left lingual nerve was sectioned and recovery permitted for 3 days, 3 weeks or 3 months (3 ferrets per group). A retrograde tracer, fluorogold, was injected into the damaged nerve to identify associated cell bodies in the trigeminal ganglion. Three further ferrets, receiving only tracer injection, served as uninjured controls. Indirect immunofluorescence for TRPV1 and image analysis was used to quantify the percentage area of staining (PAS) of TRPV1 in the left and right lingual nerves. Additionally, the proportion of fluorogold positive and fluorogold negative cells expressing TRPV1 in the ganglion was determined. TRPV1 expression increased significantly at the injury site of damaged nerves 3 days after injury and this was matched by a reduction in the proportion of fluorogold positive cells expressing TRPV1 in the ganglion. At 3 weeks TRPV1 expression at the injury site was still high, while in the ganglion was significantly greater than in the controls. In the 3-month recovery group TRPV1 expression in both nerve fibres and ganglion cells, was not significantly different from controls and there were no changes in expression in the fluorogold negative cells in the ganglion at any time point studied. These data suggest that after injury there is an increase in the axonal transport of TRPV1 from the cell bodies to the damaged axons and this is followed by an increase in synthesis in the ganglion. These changes in expression may be involved in development of sensory disturbances or dysaesthesia after injury.     

Short-term dietary phosphate restriction up-regulates ileal fibroblast growth factor 15 gene expression in mice

Members of the fibroblast growth factor (FGF) 19 subfamily, including FGF23, FGF15/19, and FGF21, have a role as endocrine factors which influence the metabolism of inorganic phosphate (Pi) and vitamin D, bile acid, and energy. It has been reported that dietary Pi regulates circulating FGF23. In this study, the short-term effects of dietary Pi restriction on the expression of FGF19 subfamily members in mice were analyzed. An initial analysis confirmed plasma FGF23 levels positively correlated with the amount of dietary Pi. On the other hand, ileal Fgf15 gene expression, but not hepatic Fgf21 gene expression, was up-regulated by dietary Pi restriction. In addition, we observed the increase of plasma 1,25-dihydroxyvitamin D [1,25(OH)₂D] levels by dietary Pi restriction, and the up-regulation of ileal Fgf15 mRNA expression by 1,25(OH)₂D₃ and vitamin D receptor (VDR). Importantly, dietary Pi restriction-induced Fgf15 gene expression was prevented in VDR-knockout mice. Furthermore, diurnal variations of plasma triglyceride concentrations and hepatic mRNA expression of the bile acid synthesis enzyme Cyp7a1 as one of Fgf15 negative target genes was influenced by dietary Pi restriction. These results suggest that dietary Pi restriction up-regulates ileal Fgf15 gene expression through 1,25(OH)₂D₃ and VDR, and may affect hepatic bile acid homeostasis.     

Role of TRPM8 and TRPA1 for cold allodynia in patients with cold injury

Local cold injury often induces hypersensitivity to cold and cold allodynia. Sensitisation of TRPM8 or TRPA1 could be the underlying mechanisms. This was evaluated by psychophysics and axon-reflex-flare induction following topical menthol and cinnamaldehyde application in cold injury patients and healthy subjects. The patients had no signs of neuropathy except cold allodynia. We applied 20% cinnamaldehyde and 40% menthol solutions in the cold-allodynic area of the patients and in a corresponding area in healthy subjects and obtained sensory ratings during application. Thermotesting and Laser Doppler Imaging were performed before and after exposure to the compounds. Menthol did not induce axon-reflex-erythema in patients or in controls. After menthol cold pain threshold was decreased in healthy subjects; however, no further sensitisation was observed in the patients moreover in some patients an amelioration of their cold allodynia was observed. Cinnamaldehyde-induced pain sensation did not differ between patients and controls. Heat pain thresholds following cinnamaldehyde were lowered to a similar extent in patients and controls (43-39.8 and 44-39 degrees C) and also the axon-reflex-flare responses were comparable. No evidence for sensitisation of responses to TRPM8 or TRPA1-stimulation was found in patients with cold injury-induced cold allodynia. The lack of TRPM8 induced axon-reflex indicates that also de-novo expression of TRPM8 on mechano-insensitive C-nociceptors does not underlie cold allodynia in these patients. We conclude from these data that the mechanisms for the induction of cold allodynia in the patients with cold injury are independent of TRPM8 or TRPA1 and differ therefore from neuropathic pain patients.     

Oral and cutaneous thermosensory profile of selective TRPV1 inhibition by ABT-102 in a randomized healthy volunteer trial

The capsaicin receptor (TRPV1) antagonist ABT-102 demonstrates efficacy in multiple preclinical pain models. However, evolving clinical data for this compound class suggest potentially profound drug-induced thermosensory impairment. Safety and tolerability of ABT-102 were assessed in a multiple-dose, double-blind, placebo-controlled, randomized healthy volunteer trial. Thirty-six participants were randomized in a 2:1 ratio to ABT-102:placebo in 3 dose groups (1 mg, 2 mg, and 4 mg twice a day) and confined to an inpatient research unit for a 7-day treatment period and 3 follow-up days. Outcome measures included: oral and cutaneous cold detection, warm detection (WDT), and heat pain thresholds (HPT); oral perceived heat intensity (oral liquid test); time to hand withdrawal (water bath test); and cutaneous pain intensity (long thermal stimulus). Significant dose-dependent (placebo- and baseline-adjusted) increases in HPT and reduced painfulness of suprathreshold heat were present from days 1-7. For ABT-102 4 mg twice a day, model-based mean differences from placebo (95% confidence interval) were as follows: oral HPT, day 1 = 2.5°C (0.6-4.4), day 5 = 4.4°C (2.5-6.3); cutaneous HPT, day 2 = 3.3°C (1.4-5.3), day 5 = 5.3°C (3.3-7.2); oral WDT, day 1 = 2.6°C (0.5-4.7), day 5 = 2.7°C (0.6-4.9); cutaneous WDT, day 2 = 1.3 (0.0-2.6), day 5 = 1.6 (0.3-2.8) (all P < 0.05). Oral liquid test and water bath test results followed a similar pattern. There was no effect on cutaneous cold detection. All effects were fully reversed by day 10. There were no other relevant safety findings. Core body temperature remained below 39°C in all participants. In conclusion, ABT-102 potently and reversibly increased HPT and reduced painfulness of suprathreshold oral/cutaneous heat.     

Exenatide upregulates gene expression of glucagon‐like peptide‐1 receptor and nerve growth factor in streptozotocin/nicotinamide‐induced diabetic mice

Glucagon‐like peptide‐1 (GLP‐1) is an incretin hormone that has modulating effects on insulin release. GLP‐1 and receptors for GLP‐1 are widely expressed throughout the body including the brain. The expression of GLP‐1 receptors is very specific to large neurons in hippocampus, neocortex, and cerebellum. GLP‐1 receptor stimulation enhances glucose‐dependent insulin secretion and lowers blood glucose in type 2 diabetes mellitus. Studies on adipobiology of neurotrophins have focused on nerve growth factor (NGF) as an example of adipose‐derived neurotrophins. Compromised trophic factor signaling may underlie neurodegenerative diseases ranging from Alzheimer's disease to diabetic neuropathies. Exenatide, a potent and selective agonist for the GLP‐1 receptor, is currently approved for the treatment of type 2 diabetes mellitus. The aim of this study was to assess the effect of chronic exenatide treatment on the hippocampal gene expression levels of GLP‐1 receptor and NGF in diabetic mice. The effects of chronic exenatide treatment (0.1 μg/kg, s.c., twice daily for 2 weeks) on GLP‐1 receptor and NGF gene expression levels in the hippocampus of streptozotocin/nicotinamide (STZ–NA)‐induced diabetic mice were assessed by quantitative real‐time polymerase chain reaction (RT‐PCR). The results of this study revealed that hippocampal gene expression of GLP‐1 receptor and NGF were downregulated in diabetic mice. Importantly, a significant increase in the gene expression level of GLP‐1 receptor and NGF was determined after 2 weeks of exenatide administration. Increased gene expression level of GLP‐1 receptor and NGF may underlie the beneficial action of exenatide in STZ/NA‐induced diabetes.     

双醋瑞因干预体外培养关节软骨结缔组织生长因子的表达

背景:在骨关节炎软骨退变中结缔组织生长因子作为一种重要的效应分子在软骨细胞的增殖、分化方面发挥重要作用。临床应用双醋瑞因治疗骨关节炎已取得了良好的效果,但其治疗的确切机制尚不清楚。目的:观察不同浓度双醋瑞因对体外白细胞介素1β诱导下软骨细胞中结缔组织生长因子的影响。方法:体外培养SD大鼠关节软骨细胞,重组人白细胞介素1β刺激软骨细胞制备体外骨关节炎模型。实验分组:正常对照组不给予任何处理因素;模型对照组给予重组人白细胞介素1β;实验组给予不同浓度双醋瑞因+10μg/L重组人白细胞介素1β。利用MTT比色法观察软骨细胞的增殖情况,Western Blot法检测结缔组织生长因子的表达。以上实验均重复3次。结果与结论:MTT结果显示,与正常对照组比较,双醋瑞因能促进软骨细胞MTT增殖活性,以浓度为10-5 mol/L更明显(P<0.01),白细胞介素1β作用后各实验组软骨细胞增殖能力下降(P<0.05);但与正常对照组比较,无论是否加白细胞介素1β,浓度为10-4 mol/L和10-5 mol/L双醋瑞因仍能促进软骨细胞MTT增殖活性(P<0.05)。Western Blot检测结果显示,白细胞介素1β能够降低结缔组织生长因子的表达(P<0.01),浓度为10-5 mol/L双醋瑞因能够显著促进白细胞介素1β诱导下结缔组织生长因子的表达,显著高于模型对照组(P<0.01)。提示双醋瑞因可以促进白细胞介素1β诱导下结缔组织生长因子的高表达,其可能是双醋瑞因促进软骨细胞的分化增殖,治疗骨关节炎的作用机制之一。     

碱性成纤维细胞生长因子在胚胎卵黄囊造血过程中的表达

目的 探讨人胚胎发育过程中碱性成纤维细胞生长因子1(FGF1)对造血细胞形成的影响,研究FGF1、血管内皮生长因子受体(KDR)、CD133、CD34和转录凶子Ihh、SCL、GATA-1、GATA-2和PU.1在卵黄囊中的表达,了解FGF1、造血细胞和转录因子在胚胎造血过程中的作用和关系.方法 采用药物流产孕3～12周人胚胎,冰冻切片,免疫组织化学SP染色,并应用分析软件进行分析,同时进行造血相关转录因子RT-PCR扩增分析.结果 孕3～12周人胚卵黄囊血岛均由外周的血管内皮细胞和中心的造血细胞组成.在16 d的卵黄囊中脏壁中胚层嗣血岛周边表达FGF1阳性产物,而血岛内少有FGF1表达,低表达KDR,不表达CD34、CD133;21 d上述抗体均有表达,灰度值减少,染色增强;30d时在血岛和中胚层发现强染色CD34+、CD133+和KDR+细胞,灰度值分别由156±16、173±18和160±14降低为53±7、52±6和69±8;FGF1呈强阳性表达,灰度值由161±13急降至40±5,达到最低值.有些阳性细胞沿血岛边缘形成血管样结构.45 d时中等程度表达CD34、CD133、KDR细胞数量增多,细胞聚集成团分布于血岛中;FGF1阳性细胞也多聚集分布在血岛中,中胚层少有分布,灰度值增加.7周后CD133+、KDR+、CD34+细胞明显减少,灰度值增加,染色变浅,卵黄囊弱表达FGF1,灰度值增加为179±22.RT-PCR结果显示,不同时间的卵黄囊均表达Ihh、SCL、GATA-1和GATA-2,PU.1在16 d不表达,此后表达.结论 卵黄囊造血细胞的发育受FGF1和转录因子的诱导和调节,提示FGF1介导的信号通路对造血中胚层模式的形成、成血管血液干细胞的扩增以及造血十细胞的动态平衡具有重要意义.FGF通路可能通过调节GATA1等转录因子表达水平和活性来调控卵黄囊造血.     

子痫前期患者胎盘组织及外周血中胎盘生长因子及可溶性血管内皮生长因子受体1的表达及意义

目的 研究子痫前期患者胎盘生长因子(placental growth factor, PlGF)、可溶性血管内皮生长因子受体1(soluble vascular endothelial growth factor receptor 1, sFlt-1)的mRNA及蛋白质在胎盘组织中的表达及外周血中的浓度水平, 探讨其与子痫前期的关系。 方法 选取57例子痫前期患者（轻度子痫前期31例、重度子痫前期26例）和60例健康孕妇,用半定量RT-PCR及western blot分别检测胎盘组织中sFlt-1、PlGF的mRNA及蛋白质的表达水平, ELISA法检测血清中PlGF和sFlt-1水平。 结果 轻、重度子痫前期患者组胎盘组织PlGF mRNA、蛋白质表达水平以及血清PlGF浓度均显著低于健康孕妇组（t分别为14.22、21.80、12.10、15.17、7.14、10.18,P均<0.01）;而胎盘组织sFlt-1 mRNA、蛋白质表达水平以及血清sFlt-1浓度显著高于健康孕妇组（t分别为12.43、17.06、13.70、18.84、13.55、15.19,P均<0.01）;轻、重度子痫前期组外周血PlGF和sFlt-1之间呈显著负相关（r=-0.49, r=-0.53,P<0.05）。 结论 子痫前期胎盘组织中PlGF水平降低伴sFlt1水平升高与子痫前期的发病密切相关。     

肥厚腰椎黄韧带中成纤维细胞生长因子、转化生长因子及结缔组织生长因子的表达

背景：腰椎黄韧带肥厚是临床上引起腰椎管狭窄的主要因素之一,但其分子机制仍不是非常清楚。目的：分析纤维化相关细胞因子碱性成纤维细胞生长因子、转化生长因子β1和结缔组织生长因子在腰椎黄韧带肥厚过程中的作用。方法：取临床手术所取黄韧带,对照组6例（椎管内占位且无腰椎不稳患者黄韧带）、突出组（单纯腰椎间盘突出症患者黄韧带）6例、腰椎管狭窄症组6例。采用实时定量RT-PCR的方法检测各组黄韧带中碱性成纤维细胞生长因子、转化生长因子β1、结缔组织生长因子及Ⅰ、Ⅲ、Ⅴ型胶原蛋白的mRNA含量,分析3个细胞因子在黄韧带肥厚过程中的作用。结果与结论：腰椎管狭窄症组碱性成纤维细胞生长因子mRNA表达均明显高于突出组和对照组（均P 〈0.05）;腰椎管狭窄症组转化生长因子β1mRNA在3组中的表达明显高于对照组和突出组（均P 〈0.01）;结缔组织生长因子 mRNA 3组间差异无显著性意义（P 〉0.05）。腰椎管狭窄症组Ⅰ型胶原蛋白mRNA表达明显高于突出组和对照组（均P 〈0.05）;Ⅲ型胶原蛋白、Ⅴ型胶原蛋白mRNA表达3组之间差异无显著性意义（P 〉0.05）。结果说明碱性成纤维细胞生长因子、转化生长因子β1在腰椎黄韧带肥厚形成过程中有重要作用,引起黄韧带肥厚的主要胶原产物为Ⅰ型胶原蛋白。     

血小板源生长因子B链的纯合二聚体与血管内皮细胞生长因子在血管网织细胞瘤中的表达及临床意义

目的研究血小板源生长因子B链的纯合二聚体（PDGF—BB）与血管内皮细胞生长因子（VEGF）在正常脑组织和血管网织细胞瘤中的表达及PDGF—BB与VEGF之间的相关性。方法应用免疫组织化学链霉菌抗生物素蛋白-过氧化物酶连接（SP）法对32例人血管网织细胞瘤和10例正常脑组织中PDGF—BB和VEGF的表达进行检测和统计学分析。结果血管网织细胞瘤组织中PDGF—BB和VEGF高于正常脑组织中PDGF—BB和VEGF表达,PDGF-BB灰度值为101．2±15．7VS180．2±14．6,VEGF灰度值为104．4±15．8VS181．5±14．6（均P〈0．01）,并且血管网织细胞瘤组织中PDGF—BB与VEGF表达呈正相关（r＝0．842,P〈0．01）。结论血管网织细胞瘤可分泌PDGF—BB和VEGF,这两种因子在作用机制上可能有密切的联系或存在因果关系。检测PDGF—BB和VEGF。可以为血管网织细胞瘤的发病机制研究及临床治疗提供新的思路。抗PDGF-BB及抗VEGF联合治疗可能成为治疗血管网织细胞瘤的新方法。