角质细胞生长因子促人角膜上皮细胞生长的研究

目的寻找促进角膜上皮损伤修复的有效方法。方法用３Ｈ胸腺嘧啶核苷（３ＨＴｄＲ）掺入及液体闪烁技术，观察角质细胞生长因子（ＫＧＦ）对体外培养的人角膜上皮细胞ＤＮＡ合成的影响，并计算细胞倍增时间。结果１～１００ｎｇ／ｍｌＫＧＦ有明显促进人角膜上皮细胞ＤＮＡ合成的作用，且呈剂量依赖性（ｒ＝０．９２３３，Ｐ＜０．００１）。１０ｎｇ／ｍｌＫＧＦ明显缩短了细胞倍增时间（３１．５９±４．８８ｈ，与对照组４０．９８±５．２０ｈ比较，Ｐ＜０．０５）。结论外源性ＫＧＦ对体外培养的人角膜上皮细胞有明显的促细胞增生作用。表明ＫＧＦ具有应用于临床，促角膜上皮损伤修复的可能性。     

表皮生长因子对晶状体和角膜上皮细胞增生的刺激作用

目的探讨不同浓度的表皮生长因子（ＥＧＦ）对晶状体和角膜上皮细胞增生的刺激作用。方法利用培养的兔晶状体和角膜上皮细胞，通过ＭＴＴ方法，检测细胞增殖密度。培养晶状体上皮细胞的ＥＧＦ浓度（１～２５０ｎｇ／ｍｌ）分为８组；而培养角膜上皮细胞的ＥＧＦ浓度（４～１０００ｎｇ／ｍｌ）分为１０组。结果ＥＧＦ浓度在３２ｎｇ／ｍｌ时，促晶状体上皮细胞的增生作用最强，与无血清组比较差异非常显著（Ｐ＜０．０１）。促角膜上皮细胞增生的最佳浓度为１６ｎｇ／ｍｌ，与无血清组比较差异显著（Ｐ＜０．０５）。结论上述结果为今后在细胞和分子水平上研究白内障和角膜伤口愈合的发生规律及其机制提供实验资料。     

异搏定对培养角膜基质细胞分泌转移生长因子—β1的影响

目的 探讨异搏定对体外培养兔角膜基质细胞分泌ＴＧＦ－β１的影响。为预防或减轻ＰＲＫ后混浊形成提供线索。方法 将传代细胞，在加药培养后４８ｈ，应用ＴＧＦ－β１Ｆｍａｘ＾ＴＭＩｍｍｕｎｏＡｓｓａｙＳｙｓｔｅｍ试剂盒和培养上清中ＴＧＦ－β１含量进行检测，根据标准曲线分别计算出各孔ＴＧＦ－β１的浓度。结果 Ｖｅｒａｐａｍｉｌ浓度５μｇ／ｍｌ和１０μｇ／ｍｌ，作用４８ｈ，培养上清中ＴＧＦ－μ１含量明显降低。     

激光角膜光学切除术后兔角膜上皮细胞凋亡的研究

目的 探讨激光角膜光学切除术后（ｐｈｏｔｏｒｅｆｒａｃｔｉｖｅ ｋｅｒａｔｅｃｔｏｍｙ,ＰＲＫ）伤口愈合过程中角膜上皮细胞凋亡情况。方法 对６只兔双眼分别行Ｐ．Ｒ,激光切削参数为一８．０Ｄ、１５６μｍ深,消隔直径５．６ｍｍ。将兔分别于ＰＲＫ后１ｍｏ、３ｍｏ处死,取下角膜进行冰冻切片,用的位标记检测法（ＴＵＮＥＬ法）分别检测角膜上皮细胞凋亡情况。结果 正常兔角膜可见上皮上皮浅层有少量细胞亡,ＰＲＫ后     

人表皮生长因子对体外培养兔角膜内皮细胞影响的实验研究

目的：明确人表皮生长因子（ｈｕｍａｎ Ｅｐｉｄｅｒｍａｌ Ｇｒｏｗｔｈ Ｆａｃｔｏｒ,ｈＥＧＦ）对角膜内皮细胞的影响。方法：采用体外培养的兔角膜内皮细胞,观察接种后不同时点ｈＥＧＦ对其生长状态的影响;兔角膜片内皮损伤模型,离体培养后行Ｈ－Ｅ染色和Ｈ＾３－ＴｄＲ掺入放射自显影,观察ｈＥＧＦ对其修复的影响。结果：兔角膜内皮细胞体外培养ｈＥＧＦ组细胞数高于对照组,并使细胞形态产生纺锤形改变;角膜内皮细胞     

抗增生药物对人视网膜神经胶质细胞的抑制作用

目的寻找抑制视网膜、玻璃体内细胞增生的有效药物;明确抗增生药物秋水仙碱、道诺霉素和５－氟尿嘧啶（５－Ｆｕ）对体外培养的人视网膜胶质（ｒｅｔｉｎａｌ ｇｌｉａ,ＲＧ）细胞的作用。方法用 ＭＴＴ法测定秋水仙碱（０．５～１６．０μｇ／ｍｌ）、道诺霉素（０．１～３．２μｇ／ｍｌ）和５－Ｆｕ（０．５～１６．０μｇ／ｍｌ）对体外培养人ＲＧ细胞的作用。结果秋水仙碱（１．０～１６．０μｇ／ｍｌ）、道诺霉素（０．２～３．２０μｇ／ｍｌ）和 ５－Ｆｕ（１．０～１６．０μｇ／ｍｌ）３组药物对培养细胞均有抑制作用,与对照组均差别显著（Ｐ＜０．０１）,ＩＤ_（５０）分别为３．１１μｇ／ｍｌ,０．７９μｇ／ｍｌ和５．２３μｇ／ｍｌ。结论秋水仙碱、道诺霉素和５－Ｆｕ对ＲＧ细胞有明显抑制作用。     

碱性成纤维细胞生长因子促人晶状体上皮细胞增生的研究

目的 探讨碱性成纤维细胞生长因子（ｂ－ＦＧＦ）在后发性白内障形成过程中的作用。方法 应用体外培养人的晶状体上皮细胞，第３代细胞悬液接种于２４孔培养板内，加入不同终浓度的ｂ－ＦＧＦ（０，１，０，１０．０，１００．０ｎｇ／ｍｌ），每组重复３孔，培养７２ｈ后，细胞计数。结果 ｂ－ＦＧＦ添加组，细胞增生呈浓度依赖性，ｂ－ＦＧＦ浓度越高，细胞增生越快。结论 白内障手术后房水中内源性ｂ－ＦＧＦ的增加可能是后发     

抗增生药物对人视网膜神经胶质细胞伯抑制作用

目的 寻找抑制视网膜、玻璃体内细胞增生的有效药物;明确抗增生药物秋水仙碱、道诺霉素和５－氟尿嘧啶（５－Ｆｕ）对体外培养的人视网胶质（ｒｅｔｉｎａｌ ｇｌｉａ,ＲＧ）细胞的作用。方法 用ＭＴＴ法测定秋水仙碱（０．５～１６．０ｕｇ／ｍｌ）、道诺霉素（０．１～３．２ｕｇ／ｍｌ）和５－Ｆｕ（０．５～１６．０ｕｇ／ｍｌ）对体外２人ＲＧ细胞的作用。结果 秋水仙碱（１．０～１６．０ｕｇ／ｍｌ）、道诺霉素（０．２     

基因重组人表皮生长因子治疗兔角膜损伤的研究

目的 探讨基因重组人表皮生长因子（ｒｈＥＧＦ）对治疗角膜损伤的疗效。方法 新西兰白兔２４只,全麻下切除角膜前板层,直径８ｍｍ,约１／３角膜厚度。将其随机均分为４组,每组６只（１２只眼）,分别滴１、１０、１００μｇ／ｍｌ ｒｈＥＧＦ液,对照组滴生理盐水,每天４次,共７天。用计算机图像处理测量角膜损伤面积。结果 １、１０、１００μｇ／ｍｌ ｒｈＥＧＦ治疗组角膜上皮愈合速率分别为９．３１、９．９６、９．     

角膜缘干细胞缺乏对角膜上皮损伤修复的影响

目的探讨角膜缘干细胞的功能。方法采用生物医学图像分析系统,计算切除角膜缘上皮组织的兔眼的角膜上皮愈合速率;用光学显微镜观察角膜上皮损伤修复过程的组织学变化。结果在角膜上皮损伤８～３２小时内,实验组与对照组角膜上皮愈合速率分别为１．２２±０．１９ｍｍ２／ｈ和１．４５±０．２０ｍｍ２／ｈ（ｔ＝３．８９,Ｐ＜０．０１）,实验组角膜上皮损伤愈合速度明显比对照组慢。实验组角膜可见大量的杯状细胞和新生血管,并随时间而逐渐向中央区发展。结论实验结果进一步证实了角膜上皮干细胞存在于角膜缘,角膜缘干细胞缺失,可使角膜上皮增殖能力丧失,角膜缘屏障功能下降,导致持续性角膜上皮糜烂、结膜组织长入和新生血管形成。     

EGF联合KGF促进人角膜上皮细胞生长

目的:寻找促进角膜上皮损伤修复,治疗持续性角膜上皮缺损的有效药物方法:用~3H—胸腺嘧啶核苷(~3H—TDR)掺入及液体闪烁技术,观察外源性表皮生长因子(EGF)联合角蛋白细胞生长因子(KGF)对体外培养的角膜上皮细胞DNA合成的影响,并计算细胞倍增时间。结果:10ng/mlEGF,10ng/mlKGF单独或联合应用均有明显促进人角膜上皮细胞DNA合成的作用(与对照组比较 P<0.01),联合用药,作用更强(P<0.05)。应用EGF与KGF明显缩短了细胞倍增时间。结论:外源性EGF与KGF对体外培养的人角膜上皮细胞有明显的促细胞增生作用,联合用药,效果更佳。表明EGF与KGF具有应用于临床,促进角膜上皮损伤修复的可能性。眼科学报1996; 12:107-109。     

血小板活化因子致角膜上皮伤口迟愈合的实验研究

目的　利用兔去上皮角膜模型 ,研究血小板活化因子 (PAF)对角膜伤口愈合的作用及其分子生物学机制。方法　离体角膜上做正中直径 7mm圆形去上皮角膜伤口。去上皮角膜分为 3组 ,即对照、PAF及BN (PAF拮抗剂 )组 ,培养 4 8h后 ,行角膜上皮染色观察伤口愈合状况。分别对兔角膜上皮 (RCE)和角膜基质 (RCK)细胞进行体外传代培养 ,RCE和RCK细胞经PAF和 (或 )BN处理 ,培养 2 4h ,提纯RNA。应用RT PCR及核酸杂交技术分别检测肝细胞生长因子 (HGF)、角质形成生长因子 (KGF)及表皮生长因子 (EGF)基因在RCK和RCE细胞及HGF受体 (HGF R)基因在RCE细胞中的表达强度。分别应用CyQUANT荧光结合和Boyden小房技术检测PAF对RCE细胞黏附、增殖和迁徙的影响。结果　PAF (10 0nmol/L )明显抑制角膜上皮伤口愈合 ,4 8h对照、PAF和BN组角膜上皮未愈合面积经电脑图像分析分别为 :(6 0± 1.5 )U、(5 8 0± 7 0 )U和 (5 0± 1 0 )U。PAF明显增强RCE细胞黏附作用 ,对照、PAF和BN组每 96孔板贴附细胞数荧光光度平均值分别为 :36 96± 372、790 8± 6 71和 3487± 32 4。RT PCR结果显示 :PAF使HGFmRNA在RCK的表达强度降低 4 .1倍 ,同时明显减弱HGF R在RCE细胞中的表达 ,核酸杂交实验证实PCR结果。结论PAF明显增强RCE细胞的黏附作用 ,     

The mechanism of accelerated corneal epithelial healing by human epidermal growth factor

The effect of biosynthetic human epidermal growth factor (hEGF) was investigated on a 10-mm diameter corneal epithelial defect model in rabbits. Topical application of over 10 micrograms/ml of hEGF five times a day significantly enhanced the epithelial healing rate, in a dose-dependent manner. The maximum healing rate was observed in eyes treated with 20 micrograms/ml of hEGF (1.59 +/- 0.26 mm2/h), whereas application of less than 5 micrograms/ml of hEGF did not increase the rate of epithelial regeneration compared statistically with control vehicles (1.03 +/- 0.24 mm2/h). S-phase analysis indicated that hEGF treatment induced a high rate of epithelial replication, particularly near the limbal region, during 12 to approximately 24 hours after wounding, followed by massive cell replication from 1 mm behind the leading edge through the limbus during 24-48 hours. The change in number and distribution of S-phase cells thereafter did not essentially differ between hEGF-treated and control groups. In concordance with the S-phase analysis, there was a statistically significant increase in the DNA content in regenerating epithelium at 48 and 72 hours in the hEGF-treated group. These findings indicate that hEGF-induced acceleration of large corneal epithelial wound healing is associated with about twofold cell replication in the regenerating epithelium during 24 to approximately 48 hours after wounding. It is concluded that cell proliferation induced by hEGF, particularly that in limbal and peripheral corneal epithelial cells, may play an important role in accelerating epithelial healing.     

Keratinocyte Growth Factor-2 on the Proliferation of Corneal Epithelial Stem Cells in Rabbit Alkali Burned Cornea

Purpose: To determine whether the topical application of keratinocyte growth factor-2 (KGF-2) can enhance corneal epithelial healing in rabbit alkali burned cornea. In addition, the distribution and proliferation of corneal epithelial stem cells in KGF-2-treated and control corneas were investigated to explain their mechanisms of effects on the epithelium. Methods: Twenty-four New Zealand eyes were divided into four groups, treated with KGF-2 solution (1, 50, 100 μg/ml) and PBS solution. Eighth millimeter filter paper discs, produced by standard paper punch, were soaked for 15 sec in 0.5N NaOH solution. The alkali-soaked discs were applied to the central cornea, centered on the pupil and held gently in position with forceps for 1 min. The cornea was finally irrigated over 1 min with 100 ml balanced salt solution (BSS). Keratinocyte growth factor-2 was then applied topically three times a day. The phosphate-buffered saline (PBS) group was served as a control. Each corneal epithelial defect was subsequently photographed every 24 hours with a slit lamp and was measured by computer-assisted digitizer. In each group, two rabbits were sacrificed for light microscopic examination after the interval of 7, 14 and 21 days. Meanwhile, the cornea epithelium was examined by immunohistochemistry for P63, AE5, EGFR. Results: Topical application of 10 μg/ml to 100 μg/ml KGF-2 significantly accelerated corneal epithelial wound healing when compared with controls. After 24 hours, epithelial healing rate of the 100 μg/ml KGF-2 group and the PBS treated group was (74±6)% and (40±8)% (P < 0.05). After 48 hours, the rate of the C group was (94±6)%, whereas in the control group it was (73±12)% (P < 0.05). Epithelial defects were often recurrent, which happened only two times in the 100 μg/ml KGF-2-treated group, but many times in the control group. In the corneal epithelial stem cell analysis, the number of the P63 positive cells was higher in the KGF-2-treated corneal epithelium than in the controls. The P63 positive cells in the alkali burned epithelium were found not only in the limbal area but also in the central cornea. In addition, the number of stem cells in each group came to the maximum on the 14th day. For example, on the 7th day after alkali injury, it was 40.3±2.1 NPC in the non-limbal area of 50 μg/ml KGF-2-treated group; whereas, it was 84.8±2.7 NPC on the 14th day(P = 0.000). Conclusions: From the daily evaluation of the corneal surface as well as the microscopic examinations at the end of the three periods of observation, we concluded that KGF-2 provided a beneficial effect in the treatment of alkali burns of the cornea. Furthermore, the results of epithelial stem cell analysis demonstrated that KGF-2 accelerated the corneal epithelial healing by markedly stimulating epithelial stem cells proliferation and making them migrate to the central cornea.     

Hepatocyte growth factor and keratinocyte growth factor regulation of epithelial and stromal corneal wound healing

PURPOSE: To investigate the effects of hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) on early wound healing in the corneal epithelium and stroma. SETTING: Cell and Molecular Biology Unit, Department of Optometry and Vision Sciences, Cardiff University, and the Cardiff Institute of Tissue Engineering and Repair, Cardiff, United Kingdom. METHODS: Corneal keratocyte cell cultures and wounded corneal organ cultures (both maintained in serum-free conditions) were treated with 0.1 to 100 ng/mL of HGF or KGF for up to 5 days. Cell cultures were assessed for proliferation, migration, and differentiation into myofibroblasts. Organ cultures were used to evaluate the effect of HGF and KGF on reepithelialization following a wound, epithelial morphology and stratification, keratocyte numbers directly beneath the wounded area, and differentiation into myofibroblasts. RESULTS: The 2 growth factors had opposite effects on the rate of reepithelialization, with HGF delaying and KGF accelerating epithelial coverage of the wound. Morphologic assessment showed that both growth factors affected the stratification and differentiation of the epithelium. Both factors stimulated proliferation of keratocytes in serum-free cell culture, although neither induced the appearance of myofibroblasts. This was in contrast to wounded organ cultures treated with 100 ng/mL HGF, in which large numbers of myofibroblasts were observed under the wound. Control corneas and those receiving KGF contained very few myofibroblasts. Keratocyte repopulation of the denuded area under the wound was enhanced in the presence of HGF but decreased in response to KGF. CONCLUSIONS: Hepatocyte growth factor and KGF appeared to have potent and often opposite effects on epithelial and stromal cells following a wound. Hepatocyte growth factor was more detrimental than KGF, resulting in an aberrant epithelium and mass differentiation of keratocytes into myofibroblasts. Inhibition of HGF may be an appropriate therapeutic intervention in the case of persistent epithelial defects and to prevent fibrosis following a corneal stromal wound such as can occur after refractive surgery.     

EGF does not enhance corneal epithelial cell motility

Although it is well known that epidermal growth factor (EGF) accelerates corneal epithelial wound healing by stimulating mitosis, it is also believed that EGF may directly stimulate the motility of individual corneal epithelial cells. We employed three different experimental methods to determine if EGF does indeed enhance the motility of corneal epithelial cells (independent of mitotic effects). First, the effects of EGF on the motility of tissue-cultured rat and rabbit corneal epithelial cells were investigated by a Boyden chamber assay. In rat corneal epithelium, these effects were further investigated by a second method, the agarose drop assay. Both assay techniques demonstrated no increase in corneal epithelial cell motility in the presence of EGF. These findings were corroborated by a third method which consisted of measuring the closure rate of epithelial wounds in organ-cultured rat corneas in the presence and absence of EGF, while concurrently arresting mitosis with colchicine. The wound closure rate before addition of any drug was 0.46 +/- 0.03 mm2/hr. The wound closure rate with EGF (50 ng/ml) was 0.55 +/- 0.03 mm2/hr, significantly (P less than 0.005) more rapid than the drug-free controls. However, when EGF (50 ng/ml) and colchicine (40 micrograms/ml) were used simultaneously, the acceleration of wound closure by EGF was completely negated by the presence of colchicine, resulting in a wound closure rate (0.46 +/- 0.06 mm2/hr) that did not differ significantly (P greater than 0.50) from that of the drug-free control.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphorylation of MAP kinase in corneal epithelial cells during wound healing

PURPOSE: To investigate the role of mitogen-activated protein kinase (MAPK), such as p44/42 MAPK, p38 MAPK and stress-activated protein kinase (SAPK), in corneal epithelial cells during the wound healing process. METHODS: A single non-penetrating incision was produced on rat cornea. Then the corneal wound healing process was observed with an immunocytochemical technique using specific antibodies reacting only with phosphorylated p44/42 MAPK, p38 MAPK or SAPK. Cell lysates of corneal epithelial cells in rabbits stimulated with keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF) were processed for Western blot using antibodies to phosphorylated p44/42 MAPK. RESULTS: Maximum activation of p44/42 MAPK was observed in wing and basal cells at wounded regions in rat cornea at 1 hour after the incision. Activation of p44/42 MAPK was still detected in all basal and wing cells at wounded regions at up to 24 hours when the incisions were completely closed, and then receded to normal intensity after 7 days. Neither p38 MAPK nor SAPK were activated during the wound healing process. Western blot analysis of cultured corneal epithelial cells in rabbits showed phosphorylation of p44/42 MAPK after 30 minutes in response to KGF and HGF, whereas non-activated p44/42 MAPK was ordinarily detected even at the absence of KGF or HGF. CONCLUSIONS: These results demonstrate that p44/42 MAPK is activated during the corneal wound healing process and suggest that KGF and HGF play an important role in initiation of cell migration and proliferation in the initial wound healing process by activating p44/42 MAPK.     

角质细胞生长因子2促进兔角膜碱烧伤上皮愈合机制的研究

目的　探讨角质细胞生长因子2 (KGF- 2 )对实验性兔角膜中央碱烧伤后角膜上皮愈合的作用及其机制。方法　24只新西兰白兔的24只角膜碱烧伤眼按随机数字法分成4组,每组6只眼,其中A、B、C组为治疗组,分别以3种不同浓度: 1μg/ml、50μg/ml、100μg/mlKGF 2滴眼液治疗;D组为对照组,用磷酸盐缓冲液(PBS)滴眼液治疗;观察角膜上皮愈合情况,并做形态学检查及P63、角质蛋白单克隆抗体(AE5)、表皮细胞生长因子单克隆抗体(EGFR)免疫组化研究。结果　1 ~100μg/mlKGF 2能够促进兔角膜上皮愈合。角膜碱烧伤24h后100μg/mlKGF- 2组和对照组角膜上皮愈合率分别为40%和74% (P<0 .05);第4天时4组均出现一定反复;第10天时各治疗组近完全愈合。碱烧伤后第7天即可观察到P63阳性细胞不仅存在于角膜缘区的部分基底细胞中,同时也向角膜内迁移。如角膜碱烧伤后第7天角膜缘区P63阳性细胞数: 100μg/mlKGF 2组为( 53 .8±2. 6)个,对照组为(29. 5±2. 2)个,正常角膜为(17. 0±2. 1)个(P=0. 000);同时非角膜缘区P63阳性细胞数分别为: 100μg/mlKGF 2组为(69. 5±2. 8)个,对照组为(19 5±2 8)个,正常角膜为0个(P=0 .000)。结论　KGF- 2可激活角膜上皮干细胞,使其不断增殖分化,从而促进角膜上皮损伤的愈合。     

Protein kinase C alpha and epsilon differentially modulate hepatocyte growth factor-induced epithelial proliferation and migration

Protein kinase C (PKC) isoenzymes require membrane translocation for physiological activation. We have recently shown that the growth factors such as epidermal growth factor and hepatocyte growth factor (HGF), but not keratinocyte growth factor (KGF), regulate PKCalpha activation to promote epithelial wound healing [Sharma, G.D., Ottino, P., Bazan, H.E.P., 2005. Epidermal and hepatocyte growth factors, but not keratinocyte growth factor, modulate protein kinase C alpha translocation to the plasma membrane through 15(S)-hydroxyeicosatetraenoic acid synthesis. J. Biol. Chem. 280, 7917--924]. Protein kinase C alpha (PKCalpha) and protein kinase C epsilon (PKCvarepsilon) are two differentially regulated isoenzymes. While PKCalpha requires Ca(2+) for its activation, PKEvarepsilon is Ca(2+) independent. However, growth factor-induced activation of these enzymes and their specific regulation of epithelial migration and proliferation have not been explored. In the present study, we overexpressed PKCvarepsilon fused to green fluorescent protein to examine its translocation in real-time to the plasma membrane in living human corneal epithelial cells. Stimulation with HGF and KGF demonstrated translocation of PKCvarepsilon to the plasma membrane. Because HGF activates both PKCs, this growth factor was used to stimulate wound healing. PKCalpha or PKCvarepsilon-genes were knocked down individually without affecting the basal expression of the other PKC isoforms. Gene knockdown of PKCalpha significantly inhibited HGF-stimulated proliferation of human corneal epithelial cells. In contrast, PKCvarepsilon-gene-silencing severely impaired the HGF-stimulated migratory ability of human corneal epithelial cells. When migrating epithelial cells in the cornea wound bed after injury were transfected with specific PKCalpha- or PKCvarepsilon-siRNA, there was a significant delay in wound healing. Corneal wound healing stimulated with HGF in similar conditions was also inhibited. On the other hand, overexpression of PKCalpha or PKCvarepsilon-genes fused with green fluorescent protein in migrating corneal epithelium accelerated repair of the epithelial defect. Our findings demonstrate that PKCalpha and PKCvarepsilon modulate different stages of wound healing stimulated by HGF and contribute to epithelial repair by playing selective regulatory roles in epithelial proliferation and migration, both crucial to corneal wound healing.