Experimental studies on an artificial esophagus using a collegen-silicone copolymer

An artificial esophagus consisting of a collagen-silicone copolymer prepared by special chemical and physical procedures proved to be advantageous by both physical and histological examination. The adherence to the host tissue was satisfactory when collagen was used. The esophageal prosthesis was anastomosed to the jejunum in 5 dogs and no leakage occurred during 10–30 days. In subsequent experiments, use of the artificial prosthesis in the thoracic esophagus was followed by leakage in 5 of 14 cases (35 per cent). Five of these dogs survived for over 30 days, and the longest survival was 212 days. The incidence of leakage was acceptable but stricture was a common long-term complication. Further studies are underway to refine the prosthesis, the method of anastomosis, and post-operative management.     

无血清培养人表皮细胞的研究

作者用无血清培养基培养人的表皮细胞获得成功。实验结果证明无血清培养基比含血清培养基更有利于表皮细胞生长增殖，为解决大面积烧伤救治中皮源不足的问题提供了新的途径。     

Influence of epidermal growth factor on bovine pancreatic duct cell bicarbonate

BACKGROUND AND AIMS: Epidermal growth factor (EGF) is secreted in pancreatic juice and its receptor is expressed on pancreatic duct cells (PDCs), suggesting a physiological role which has yet to be defined. Here we examined the effects of EGF on bicarbonate production and carbonic anhydrase (CA) activity in a PDC explant model. METHODS: Bovine main PDCs were prepared and maintained in culture as explants. Levels of CA expression, phosphorylation, and enzymatic activity were measured in resting cells and compared to that of cells exposed to 10 nM secretin, 10 nM EGF, or both. Bicarbonate production was measured using the autoburette pH titration technique. RESULTS: CA protein levels were unchanged with any treatment, but enzyme activity increased by 180% with secretin treatment and was reduced by 54% with EGF. The combination treatment led to a synergistic increase 240% above basal. EGF alone did not affect bicarbonate secretion, but the normal increase observed with secretin stimulation (1.3 +/- 0.4 to 2.9 +/- 0.6 micromol/h/cm(2)) was abolished by acute EGF pretreatment. On the other hand, EGF pretreatment for 24 h significantly increased basal and stimulated secretion (2.2 +/- 0.5 and 3.8 +/- 0.5, respectively) compared to controls. CONCLUSIONS: EGF exerts a regulatory role on bicarbonate secretion by the pancreatic duct epithelium, independent of its effect on CA activity. Its inhibition of stimulated bicarbonate secretion could play a protective role in the setting of pancreatic inflammation, where increased levels of EGF are associated with reduced pancreatic juice production.     

Mitogenic and apoptotic actions of epidermal growth factor on neuroblastoma cells are concentration-dependent

BACKGROUND: In previous studies we have shown that epidermal growth factor (EGF) at concentrations between 50 and 100 ng/mL induced apoptosis in wild-type SK-N-SH neuroblastoma cells. We hypothesize that this apoptotic event separates EGF-induced neuroblastoma cell growth into a biphasic concentration-dependent process, due to activation of different signaling cascades. METHODS: Cells were incubated in concentrations of EGF ranging from 5 to 250 ng/mL for 3 days, and cell proliferation was determined by the MTT assay. Cells incubated with EGF 5, 100, or 250 ng/mL for 17 h were also assayed for apoptosis by DNA laddering. Western immunoblots were performed on whole cell lysates prepared from cells incubated with EGF (5-250 ng/mL) for 17 h. Antibodies against cleaved caspase3, p-AKT, p-GSK-3beta, p-BAD, p-RAF, p-ERK, and p-P38 were used as probes. RESULTS: A triphasic, concentration-dependent response was observed following incubation of cells with EGF. Cell proliferation was increased by EGF 5 ng/mL (P < 0.05), decreased by EGF 100 ng/mL, and increased when incubated with EGF 250 ng/mL (P < 0.05). DNA laddering only occurred after treatment with EGF 100 ng/mL. The expressions of p-ERK, p-RAF, p-BAD, and p-GSK-3beta were increased at EGF concentrations of 5-10 ng/mL. At 50-100 ng/mL EGF, the expression of cleaved caspase3 was increased. Maximal p-P38 expression was at 50 ng/mL EGF. At EGF concentrations of 150-250 ng/mL, the expressions of p-AKT and p-GSK-3beta were elevated. CONCLUSIONS: Neuroblastoma cell growth induced by EGF exhibited a triphasic pattern; cell growth was increased at EGF concentrations 5-20 and 150-250 ng/mL, but decreased at 50-100 mg/mL. Apoptosis was induced at 50-100 ng/mL EGF. Each growth phase activated different signaling molecules.     

Experimental study of an artificial esophagus using a collagen sponge, a latissimus dorsi muscle flap, and split-thickness skin

The time and effort spent trying to devise an artificial esophagus have not yet resulted in success, and leakage and strictures at the anastomotic sites remain the most frequent complications. We developed an artificial esophagus with a bilayered structure made of porous collagen sponge (artificial dermis; AD), a latissimus dorsi muscle flap (LD), and split-thickness skin (STS). We investigated whether the use of AD prevented the contraction of grafted skin and its effects on the extensibility of the neoesophagus in rabbits. We experimented with two groups. In the AD group, AD was applied to the surface of the LD. Three weeks later, the STS was grafted. In the control group, the STS was grafted directly onto the LD. The sizes of the STS in both groups 3 weeks after the graft were, respectively, 56.6% ± 4.1% and 39.0% ± 10.2% of the initial surface area of the STS (P < 0.01). The roll made in the AD group had better extensibility than that in the control group. We replaced the cervical esophagus in 12 rabbits with the neoesophagus made from AD, STS, and LD. The longest survival period was 16 days. Esophagography did not reveal either anastomotic leakage or stenosis in any of the five rabbits in the experiments. These findings suggested that AD can thus be used to create a more suitable hybrid artificial esophagus. Received: April 28, 1999 / Accepted: January 7, 2000     

纳米金对人体表皮细胞增殖作用的体外实验研究

目的：探讨纳米金对人体表皮细胞的增殖作用,为纳米金材料在皮肤组织相关医学领域的开发应用提供科学依据。方法：首先运用柠檬酸钠还原法制备纳米金溶胶,然后观察培养液中含有不同终浓度纳米金溶胶体溶液对人正常皮肤表皮细胞增殖的影响。结果：培养液中纳米金含量在0.48mg/L时就能显著促进表皮细胞增殖,随着纳米金含量增加,细胞增殖率增加,在含量达到12mg/L时,细胞增殖达到高峰,此后,细胞增殖率逐步下降。结论：培养液中纳米金含量在一定范围内具有促进人体皮肤组织表皮细胞增殖的作用。     

Sustained local application of low-dose epidermal growth factor on steroid-inhibited colonic wound healing

Background/purpose

The effects of locally administered low-dose epidermal growth factor in a steroid-inhibited wound healing were investigated in a rat model.

Methods

Long-acting release of epidermal growth factor was enabled using microspheres embedded in gelatin sponge. Study groups consisted of 60 rats with 10 in each: colonic anastomosis only (C), plus pure gelatin sponge (CG), plus epidermal growth factor loaded sponge (CE), colonic anastomosis and steroid (S), plus gelatine sponge (SG), and plus epidermal growth factor-loaded gelatine sponge (SE) groups. Bursting pressure and wound hydroxy-proline content were measured. Bursting sites were recorded. Collagen deposits, inflammation, and foreign body reactions were evaluated.

Results

Bursting pressure and hydroxy-proline contents were found lowest in the S and highest in the CE groups (P < .01). There was almost no difference between C and SE groups. Bursts were encountered in peri-anastomotic normal colon sites in the nonsteroid-treated C, CG, and CE groups. They were noted overwhelmingly at the anastomosis in steroid-inhibited S, SG, and SE groups. Histopathology results showed a standstill at the inflammatory phase of healing in S and SG groups. The best healing was observed in the CE group. Degree of collagen accumulation was well correlated with bursting pressure and hydroxy-proline content data with a negligible foreign body reaction to gelatine sponge.

Conclusions

Continuous local epidermal growth factor administration by microspheres in gelatin increases wound collagen and further enhances healing in colonic anastomoses even with steroid inhibition.     

Bladder epithelial cells from patients with interstitial cystitis produce an inhibitor of heparin-binding epidermal growth factor-like growth factor production

PURPOSE: The etiology of interstitial cystitis is unknown. We previously identified an interstitial cystitis urine factor, antiproliferative factor, that inhibits proliferation of bladder epithelial cells in vitro and complex changes in epithelial growth factor levels, including profound decreases in heparin-binding epidermal growth factor-like growth factor (HB-EGF). Bladder and renal pelvic catheterization of patients with interstitial cystitis indicated that the antiproliferative factor is made and/or activated in the distal ureter or bladder. Therefore, we determined whether bladder epithelial cells from interstitial cystitis cases produced the antiproliferative factor and whether purified antiproliferative factor could alter production of growth factors known to be abnormal in interstitial cystitis. MATERIALS AND METHODS: Antiproliferative factor activity was determined by 3H-thymidine incorporation into primary bladder epithelial cells. The antiproliferative factor was purified by size fractionation followed by sequential chromatography involving ion exchange, hydrophobic interaction and high performance liquid chromatography. HB-EGF, epidermal growth factor, insulin-like growth factor and insulin-like growth factor binding protein 3 levels were determined by enzyme-linked immunosorbent assay. RESULTS: Bladder epithelial cells from patients with interstitial cystitis produced a single antiproliferative factor with the same purification profile as that purified from interstitial cystitis urine. Purified antiproliferative factor specifically inhibited HB-EGF production by bladder epithelial cells in vitro, and the effect of interstitial cystitis urine or purified antiproliferative factor on bladder cell proliferation was inhibited by recombinant human HB-EGF in a dose dependent manner. Similar to urine HB-EGF, serum HB-EGF was also significantly lower in interstitial cystitis cases than in controls. CONCLUSIONS: Bladder epithelial abnormalities in interstitial cystitis may be caused by a negative autocrine growth factor that inhibits cell proliferation by down-regulating HB-EGF production. Furthermore, decreased levels of urine and serum HB-EGF indicate that interstitial cystitis may be a urinary tract manifestation of a systemic disorder.     

应用黑素细胞培养基在成年SD大鼠雪旺细胞培养中的实验研究

目的 寻找简单快速获得高纯度雪旺细胞的方法.方法 取20只预变性成年SD大鼠坐骨神经,经分次酶消化后,分别使用DMEM/F12(A组)和黑素细胞培养基(B组)进行培养.冷喷射(cold jet)方式传代,S-100染色鉴定.结果 采用黑素细胞培养基组的雪旺细胞纯度达到90%以上.结论 使用黑素细胞培养基及冷喷射传代操作简单,可以在短期内获得足量高纯度的雪旺细胞.     

无血清培养条件下人表皮干细胞的生物学特性研究

目的建立人表皮干细胞的无血清培养法并观察其生物学特性。方法从幼儿包皮中分离表皮细胞,用Ⅳ型胶原纯化表皮干细胞,分别在常规(血清组)、无血清(无血清1组)和无血清但添加牛垂体提取物(无血清2组)条件下进行培养。20d后观察比较3组细胞形态学变化、克隆计数、传代计数、α6及CD71表达情况,并进行细胞周期分析,检测细胞角蛋白(CK)19、CK5/8、CK10的阳性细胞百分比。结果无血清1组与血清组细胞形态相近,均可形成43个左右克隆,可传代10次,α6briCD71dim细胞百分比为(48±6)%,(72.7±6.2)%的细胞处于G0/G1期,CK19、CK5/8、CK10表达情况与血清组相近,差异无统计学意义(P>0.05)。无血清2组除细胞克隆数、CK10阳性细胞百分比高于前两组外(P<0.05),其他检测指标均偏低(P<0.05)。结论无血清培养基能选择性培养人表皮干细胞,这一模型可用于进行表皮干细胞生物学特性的相关研究。     

A trial of the in vitro sensitivity test of anti-cancer drugs using primary cultured urogenital cancer cells

Summary An in vitro chemosensitivity test was applied to clinical specimens of urogenital cancer tissues obtained at operation. Incorporation of ³H-leucine into primary cultured cells 24 h after treatment with cytotoxic drugs was used as an index for cell viability. Primary cell culture was performed using specimens obtained from 37 patients including 20 with transitional cell carcinoma, 15 with renal cell carcinoma and 2 with testicular cancer. Primary cell growth was achieved in 27 (73%) out of 37 specimens and 10 were tested for chemosensitivity. Each specimen of the tumor revealed different sensitivity to drugs, and results of quadruplicate tests for each specimen were identical. It was concluded that the present method of measuring incorporation of radioactivity using urogenital cancer cells primarily cultured in microtiter plate is practically applicable to an in vitro chemosensitivity test.     

骨髓间充质干细胞在糖尿病模型创面中向表皮细胞分化的初步研究

目的：探讨糖尿病皮肤创面微环境中的骨髓间充质干细胞分化为表皮细胞的可能性。方法：从大鼠骨髓中分离纯化骨髓间充质干细胞。选取第3—4代细胞,应用5-溴脱氧尿嘧啶（5-BrdU）标记技术进行细胞标记;同时采用一次性腹腔内注射链脲佐菌素制作糖尿病大鼠模型。2周后,在大鼠背部人工造成圆形创面,将已标记的骨髓间充质干细胞以注射方式回植入糖尿病大鼠创面组织周围,分别于注射后2、3周取材,常规石蜡包埋、连续切片,行BrdU和角蛋白免疫组织化学染色。结果：BrdU阳性细胞出现在表皮、真皮及皮下组织各层,连续切片表皮层中部分阳性细胞同时也表达角蛋白。结论：在糖尿病渍疡愈合过程中,骨髓间充质干细胞具有向表皮细胞分化的潜能。     

表皮生长因子和干细胞因子对小鼠生精细胞增殖与分化的作用

目的:观察表皮生长因子(EGF)和干细胞因子(SCF)体外促小鼠生精细胞增殖、分化的效应。方法:对7~8日龄雄性昆明小鼠生精细胞进行混合细胞体外培养,在培养液中分别添加不同浓度(5、10、20、40、100 ng/ml)的EGF和SCF,并进行EGF和SCF的交互实验,观察生精细胞的存活率和形态学变化,并对粗线期精母细胞特异性磷蛋白基因(P19)、单倍体精子细胞特异性转化蛋白基因(TP1)及染色体倍性进行检测。结果:加入EGF或SCF 2~4 d,各组均可见不同程度的细胞增殖,细胞呈团或族状,以20 ng/ml EGF组和40 ng/ml SCF组最为明显;培养第7天,单一添加20 ng/ml EGF或40 ng/ml SCF组生精细胞数和存活率显著高于与其它各组(P0.05),且40 ng/ml SCF组P19/TP1基因表达显著低于其它各组,单倍体细胞率显著高于其它各组(P0.05)。EGF与SCF配伍时,可显著增加体外培养后生精细胞数(P0.05)。结论:在混合生精细胞体外培养体系中,添加一定浓度的EGF和SCF可显著提高生精细胞数和存活率,而且SCF可提高单倍体精子的形成率;两者交互实验时,对细胞增殖有一定的叠加效应。     

重组人表皮生长因子促进大鼠皮肤创面愈合的研究

目的观察重组人表皮生长因子(rhEGF)对皮肤创面愈合的作用。方法制作大鼠背部创伤模型,采用自身平行对照,将34只大鼠背部的68个创面分成rhEGF治疗组与盐水对照组,观察大体形态和组织学改变、创面愈合时间和愈合率,测定伤后不同时间创面羟脯氨酸(OHP)含量和Ⅰ型Ⅲ型胶原比例,进行细胞DNA周期分析。结果经rhEGF治疗的创面愈合速度较盐水对照明显加快,2组平均愈合时间为(17.2±1.3)d和(20.5±1.6)d(P<0.01);外用rhEGF使创面肉芽组织生成增多,再上皮化明显,显著增加创面中OHP含量,降低Ⅰ型Ⅲ型胶原比例,加速细胞DNA复制。结论外用rhEGF可缩短创面愈合时间,增加肉芽组织及OHP含量,降低Ⅰ型Ⅲ型胶原比例,加速细胞DNA复制,明显促进皮肤创面的修复。     

Expression of the epidermal growth factor receptor family in normal and malignant urothelium

OBJECTIVE: To compare the immunohistochemically assessed expression of the epidermal growth factor receptor (EGFR) family in normal and malignant bladder urothelium, and suggest new hypotheses about their function in the development and progression of transitional cell carcinoma (TCC). PATIENTS AND METHODS: EGFR, ERBB2, ERBB3 and ERBB4 were evaluated immunohistochemically in normal urothelium (NU, 15), primary non-metastasized invasive TCC (NMC, 19) and in primary invasive TCCs with corresponding metastases (MC, 51, both specimens). RESULTS: All NU samples expressed ERBB4, none expressed ERBB2 and two expressed EGFR; all staining was uniform throughout all cell layers. ERBB2 expression increased and ERBB4 decreased from normal samples to carcinomas. There was no difference between NMCs and MCs in ERBB2, ERBB3 and ERBB4, but the NMCs expressed more EGFR than both NU and MC samples. There were no associations with T category, grade or survival. All combinations of expression levels for the four receptors were detected, with no dominant profile. CONCLUSION: We hypothesise that: (i) ERBB4 is important for differentiation in NU; (ii) ERBB2 is up-regulated with carcinogenesis in the urinary bladder but does not discriminate between bladder cancer with or without metastases; (iii) EGFR may be a marker of indolent disease. A current hypothesis, that superficial layers of NU do not express EGFR and thus protect the basal cells from the mitogenic effect of urinary EGF, is challenged.     

表皮生长因子对肾小球上皮细胞骨架的影响

目的观察阿霉素作用下体外培养的大鼠肾小球上皮细胞(GEC)通透性和细胞骨架变化并研究表皮生长因子(EGF)对它们的作用及可能途径。方法在阿霉素作用GEC24h之前给予和不给予外源性EGF，利用Milllcell—PCF Inserts检测牛血清白蛋白(BSA)滤过量，同时评价GEC细胞活力。细胞免疫荧光法和激光共聚焦显微镜观察和测定细胞骨架分子F-肌动蛋白(actin)、α-辅肌动蛋白(actinin)的表达。结果阿霉素刺激GEC24h后，BSA滤过量明显增加，而F—actin重排率增加，排列紊乱，部分解聚，胞浆内应力纤维明显减少；α-actinin趋向核周聚集。阿霉素诱导前先给予EGF，则BSA滤过量明显减少，细胞骨架恢复正常组装状态,α—actinin在胞浆内均匀分布。但在EGF之前给予EGF受体(EGFR)抑制剂AG1478或磷脂酶C(PLC)1抑制剂U73122，则EGF对阿霉素诱导下GEC的改善效应减弱，而在无EGF的阿霉素诱导之前应用AG1478或U73122，均未产生改变。结论阿霉素诱导了GEC细胞骨架的重排和破坏．致使上皮通透性增高，而EGF可能通过EGF—EGFR—PLCγ这条信号转导途径阻止了阿霉素对GEC细胞骨架的影响，保护了GEC上皮屏障。     

1064nm Q-switched Nd：YAG激光照射对表皮黑素细胞生物效应的影响

目的：探讨Q-switched Nd：YAG激光对培养状态下的人表皮黑素细胞生物效应的影响。方法：体外培养人表皮黑素细胞,用1064nmQ-switched Nd：YAG激光,光斑直径6mm,频率2HZ,分别用低能量密度与高能量密度进行照射。光学显微镜观察黑素细胞形态学变化,MTT法检测照射后0、24h、48h、72h细胞增殖活性,流式细胞仪检测照射24h后细胞周期与凋亡。结果：各能量密度组激光照射后即刻黑素细胞形态无明显改变,但24h后高能量密度照射组黑素细胞体积变小,树突数量减少,长度缩短。与对照组比较,低能量密度照射组黑素细胞增殖无显著差异,而高能量密度照射组显著降低,并在48h内逐渐下降至最低点,随后又呈明显上升趋势。与对照组相比较,低能量密度照射组S期细胞比率轻度增加,而细胞凋亡率无明显变化。高能量密度照射组S期细胞比率减少,凋亡率显著增加。结论：高能量密度Q-switchedNd：YAG激光照射对表皮黑素细胞的影响大于低能量密度,当其达到一定阈值时能抑制黑素细胞的增殖,并促进其凋亡。而这种损伤在一定范围内是可逆性的,黑素细胞会随着时间的延长进行自我修复。低能量密度激光照射对黑素细胞生物学活性的影响...     

Induction of proliferation and differentiation of cultured urothelial cells on acellular biomaterials

OBJECTIVE: To determine the optimum conditions for the proliferation of urothelial cells, leading to the confluent coverage of large surfaces of biocompatible membranes, and for their terminal differentiation. MATERIALS AND METHODS: Porcine and human urothelial cells were cultured on different matrices under different growth conditions. Proliferative activity and the viability of cells were evaluated using fluorescent markers for nuclei and cytoplasm. Growth and differentiation were assessed by histological, histochemical and immunohistochemical methods. RESULTS: Under fibroblastic induction and supplementation of 5% fetal calf serum (FCS), urothelial cells showed more proliferation than in other conditions tested. Terminal differentiation of superficial cells was achieved by lowering the concentration of FCS to 1% at the air-liquid interface. CONCLUSIONS: The mitogenic effects of the extracellular matrix content of biological membranes and fibroblastic inductive factors are synergistic with each other, and can compensate for a low FCS concentration and the absence of other additives. Lowering the FCS concentration to 1% inhibits the proliferation of urothelial cells and permits their terminal differentiation.     

Evaluation of malignancy and the prognosis of esophageal cancer based on an immunohistochemical study (p53, E-cadherin, epidermal growth factor receptor)

The subjects in this study consisted of 40 preoperative untreated esophageal squamous cell carcinoma patients. While p53 did not significantly correlate with the clinicopathological factors, E-cadherin significantly correlated with lymphatic invasion, vascular invasion, the depth of invasion, the degree of lymph node metastasis, the histological stage, and the number of lymph node metastases. Epidermal growth factor receptor (EGFR) significantly correlated with age, the depth of invasion, and the number of lymph node metastases. The 5-year cumulative survival rate was 45.7% in the p53-positive cases and 61.9% in the p53-negative cases, with no significant difference, and 87.8% in the E-cadherinpositive cases and 19.1% in the-negative cases, and the difference was significnat. The prognosis was significantly poor in EGFR-positive subjects: the 5-year survival rate was 38.6% in EGFR-positive cases and 68% in-negative cases. The 5-year survival rate in E-cadherin-negative, EGFR-positive cases was 0%, while it was 91.7% in the reverse pattern, and this difference was significant. These findings suggest that both E-cadherin and EGFR are important prognostic factors, and a more precise prognosis can thus be obtained by combining them. Such a combined technique may be very useful as an indicator for grading the biological malignancy of esophageal cancer.