人体组织反射光谱的检测研究

反射光谱是一种非侵入式的光学技术,可提高癌症实时反馈和诊断效率。本文综述了基于反射光谱技术在人体皮肤、口腔、肺、鼻咽等组织疾病的研究概况,包括离体组织和活体组织反射光谱的检测,展望了发展前景。     

Isoforms of NADP-dependent Malic Enzyme in Tissues of the Greening Maize Leaf

Scagliarini, S., Pupillo, P. and Valenti, V. 1988. Isoformsof NADP-dependent malic enzyme in tissues of the greening maizeleaf.—J. exp. Bot. 39: 1109–1119. The compartmentation of the isoforms of NADP-dependent malicenzyme (E.C. 1.1.1.40 [EC] ) has been studied in cell-free extractsand in enzymatically-isolated protoplasts of mesophyll tissue(MT) and bundle sheath (BS) strands of greening maize leaves.The etiolated leaf of 10-d-old seedlings contains a cytosolicisozyme with a pl of 5.4 ?0.1 at low specific activity (s.a,45 ? 3 nmol min^–1 mg^–1 protein), found both in MTand BS. The green leaf on the other hand contains the dominantBS chloroplast isozyme with pl 4.6 ? 0.2 at a s.a, of 370 ?40 nmol min^–1 mg^–1 protein (3.2 ? 0.5 µmolmin^–1 mg^–1 chl) and a minor, previously undescribedisoform with pl 6.5 ? 0.1 also localized in the BS at a s.a.of 38 ? 6 nmol min^–1 mg^–1 protein. Green MT protoplastshave only traces of pl 4.6 isozyme. After illumination of dark-grown seedlings, the total leaf activityshows a rapid increase (1.5-fold within 2 h), attributed mainlyto the pl 5.4 isozyme of MT protoplasts and BS strands. Thisis followed by a large increase of enzyme activity due to thecontinued rise of pl 5.4 isozyme for about 24 h and, after aninitial lag of a few hours, to the accumulation of pl 4.6 isozyme.After 18 h illumination, pl 4.6 and 5.4 isozyme activities tendto decline in the MT whereas they are still increasing in theBS, particularly the former. This pl 4.6 species has becomethe major one by 48 h illumination. The final pattern of greenleaves is established around 96 h light, when the chloroplastisozyme has attained its maximum level, the pl 5.4 isozyme ofBS strands has been superceded by the pl 6.5 species (also supposedto be cytosolic) and MT protoplasts retain little residual activity.Some metabolic implications of the changing pattern of NADP-dependentmalic isozymes during maize leaf greening are discussed. Key words: C₄, isozymes, malic enzyme, photodifferentiation, Zea mays     

人原发性肝内胆管细胞癌中HCV RNA及其NS3区抗原的分布及意义

应用原位分子杂交及免疫组分方法,使用地高辛标记ＨＣＶ５’非编码区探针及抗ＨＣＶ ＮＳ３区Ｃ３３ｃ单克隆抗体,对３５例人原发性肝内胆管细胞癌（ＰＩＣ）和癌旁肝组织的ＨＣＶ ＲＮＡ及其ＮＳ３抗原进行检测结果发现ＨＣＶ ＲＮＡ在ＰＩＣ中的阳性率为８３％,ＨＣＶ ＲＮＡ定位于癌细胞浆中,个别病例并在淋巴细胞、肝窦内皮细胞和枯否氏细胞中发现阳性信号。ＨＣＶ ＮＳ３区Ｃ３３ｃ抗原在ＰＩＣ的阳性率为８９％,阳性     

宫颈癌组织人乳头瘤病毒的荧光偏振基因分型

采用荧光偏振人乳头瘤病毒(human papillomavirus,HPV)分型新方法探讨了8种常见型别HPV在陕西宫颈癌患者中的流行情况。首先,用HPV GP5 ／GP6 通用引物PCR扩增65例早期宫颈癌(Ⅱa期内)和72例慢性宫颈炎病变组织DNA粗提物,继之将模板指导的末端延伸反应与荧光偏振检测技术结合(TDI-FP),用GP5 ／GP6 扩增区内的HPV6、11、16、18、31、33、35和58型特异性探针与PCR产物杂交后,荧光素标记的特异碱基(TAMRA-ddTTP或R110-ddGTP)在GP5 ／GP6 产物中相应的模板指导下,掺入延伸至相应探针末端,致使对应的TAMRA或R110 FP值升高,从而对扩增的HPV阳性产物进行HPV分型。65例宫颈癌患者中检出HPV57例,阳性率87．69％,72例慢性宫颈炎患者中检出HPV28例,阳性率38．89％,两组间HPV阳性率有显著性差异。宫颈癌与慢性宫颈炎患者中4种最常见的HPV型别分别是HPV 16(45．6％)、HPV 18(22．8％)、HPV 58(17．5％)、HPV 31(7．02％)和HPV 16(35．7％)、HPV 11(32．1％)、HPV 6(21．4％)、HPV 18(10．7％)。慢性宫颈炎患者中检出的HPV型别57．14％属高危型。HPV 16在两组中均最为多见。中国陕西宫颈疾病患者中HPV感染有其特点,世界范围内少见的HPV 58在陕西宫颈癌与慢性宫颈炎患者中均较为多见,在进行HPV新诊断方法及疫苗研制时应考虑到这种特点。     

人尿嘧啶糖基化酶cDNA的克隆及其在食管鳞癌组织中的检测

尿嘧啶糖基化酶是碱基切除修复过程的起始酶,对于维护基因稳定具有重要意义。在不同组织及不同细胞周期中,该酶的表达水平存在差异。通过反转录PCR克隆了人尿嘧啶糖基化酶的cDNA编码序列,进一步以克隆所得的已知UNG基因拷贝数的重组质粒作为定量标准,通过实时荧光定量RT-PCR测定了食管癌病人手术切除组织中尿嘧啶糖基化酶的mRNA水平,探讨了尿嘧啶糖基化酶表达水平与食管癌之间的联系。     

大鼠肝及脑组织中微粒体血红素加氧酶同工酶研究

采用离子交换层析和免疫印迹法分离、纯化和分析血红素和苯肼诱导后大鼠肝脏、脑组织.结果显示：纯化诱导后的大鼠肝脏,获得 HO-1和 HO-2,前者活性高于后者为2∶1.未诱导的大鼠肝脏仅获得HO-2,但诱导剂作用后,HO-1活性明显增加,而HO-2未见改变.HO-1和HO-2表观分子质量分别为30 ku和36 ku.诱导剂未作用的肝脏及作用的脑层析后仅获得HO-2活性的洗脱峰.免疫印迹法检测发现大鼠肝脏HO-2抗体与脑HO-2间有交叉反应,与肝脏HO-1无反应.实验表明在诱导剂作用的大鼠肝脏内含HO-1和HO-2同工酶,其中HO-1为诱导型酶.诱导剂作用的脑仅含HO-2.两种构型在表观分子质量,诱导性和免疫化学特性方面明显不同.     

Relative Expression of mRNAS Coding for Glutaminase Isoforms in CNS Tissues and CNS Tumors

Glutaminase (GA) in mammalian tissues occurs in three isoforms: LGA (liver-type), KGA (kidney-type) and GAC (a KGA variant). Our previous study showed that human malignant gliomas (WHO grades III and IV) lack expression of LGA mRNA but are enriched in GAC mRNA relative to KGA mRNA. Here we analyzed the expression of mRNAs coding for the three isoforms in the biopsy material derived from other central nervous system tumors of WHO grades I–III. Non-neoplastic resective epileptic surgery samples served as control, as did cultured rat astrocytes and neurons. The GAC mRNA/KGA mRNA expression ratio was as a rule higher in the neoplastic than in control tissues, irrespective of the cell type dominating in the tumor or tumor malignancy. LGA mRNA expression was relatively very low in cultured astrocytes, and very low to absent in astrocytoma pilocyticum, ependymoma and subependymal giant cell astrocytoma (SEGA), tumors of astrocytic origin. LGA mRNA expression was almost as high as that of KGA and GAC mRNA in cultured neurons and epileptic surgery samples which were enriched in neurons. LGA mRNA was also relatively high in ganglioglioma which contains a discernable proportion of neuronal cells, and in oligodendroglioma. The results show that low expression of LGA mRNA is a feature common to normal astrocytes and astroglia-derived tumor cells or ependymomas and can be considered as a cell-type, rather than a malignancy marker.     

Cadmium Concentration in Human Autopsy Tissues

The concentration of cadmium in human tissues obtained on the basis of autopsies of non-poisoned Polish people (n = 150), aged from 1 to 80 years, examined between 1990 and 2010, is presented. The following values were found in wet digested samples by flame atomic absorption spectrometry (FAAS) (mean ± SD, median, and range, μg/g of wet weight): brain 0.020 ± 0.031, 0.084, 0–0.120 (n = 41); stomach 0.148 ± 0.195, 0.084, 0–1.25 (n = 89); small intestine 0.227 ± 0.231, 0.130, 0–0.830 (n = 39); liver 1.54 ± 1.55, 1.01, 0.015–9.65 (n = 99); kidney 16.0 ± 13.2, 14.0, 0.62–61.3 (n = 91); lung 0.304 ± 0.414, 0.130, 0–1.90 (n = 25); and heart 0.137 ± 0.107, 0.140, 0.017–0.250 (n = 4). Additionally, results (n = 13 people, aged from 2 to 83 years, 63 samples) obtained by inductively coupled plasma optical emission spectrometry (ICP OES) between 2010 and 2015 are given. The obtained data on Cd concentration in the human body can be used to estimate the amounts occurring in “healthy” people and those occurring in cases of chronic or acute poisonings with Cd compounds, which are examined for forensic purposes or to assess environmental exposure levels.     

The Histochemical Detection of iron in Tissues

The fixation and staining of iron in tissues is discussed. Procedures for demonstrating iron in hemoglobin and nuclei are also briefly considered.

Lillie's formalin buffered at neutrality gave the optimal fixation for iron. The Prussian blue method was preferred to the Turnbull blue. Lison's procedure of the former, slightly modified, gave the most satisfactory results. When a procedure is required that employs non-iron-containing reagents, Macallum's or Mallory's hema-toxylin and Quincke's ammonium sulfide method are useful. The former, though not entirely specific, is preferable under controlled conditions when the quantities of iron are small. Hemoglobin iron in paraffin sections can be demonstrated by the usual procedures for iron after previous exposure of the section to peroxide, as recommended by Brown. The property of nuclei to adsorb iron from inorganic sources and from hemoglobin can readily be shown; caution is required in interpreting the iron detected in nuclei after Macallum's sulfuric acid hydrolysis.     

Identification of Active Retinaldehyde Dehydrogenase Isoforms in the Postnatal Human Eye

Background/Objectives

Retinaldehyde dehydrogenase 2 (RALDH2) has been implicated in regulating all-trans-retinoic acid (atRA) synthesis in response to visual signals in animal models of myopia. To explore the potential role of retinaldehyde dehydrogenase (RALDH) enzymes and atRA in human postnatal ocular growth, RALDH activity, along with the distribution of RALDH1, RALDH2, and RALDH3 in the postnatal eye was determined.

Methodology

Retina, retinal pigment epithelium (RPE), choroid, and sclera were isolated from donor human eyes. RALDH catalytic activity was measured in tissue homogenates using an in vitro atRA synthesis assay together with HPLC quantification of synthesized atRA. Homogenates were compared by western blotting for RALDH1, RALDH2, and RALDH3 protein. Immunohistochemistry was used to determine RALDH1 and RALDH2 localization in posterior fundal layers of the human eye.

Principal Findings

In the postnatal human eye, RALDH catalytic activity was detected in the choroid (6.84 ± 1.20 pmol/hr/ug), RPE (5.46 ± 1.18 pmol/hr/ug), and retina (4.21 ± 1.55 pmol/hr/ug), indicating the presence of active RALDH enzymes in these tissues. RALDH2 was most abundant in the choroid and RPE, in moderate abundance in the retina, and in relatively low abundance in sclera. RALDH1 was most abundant in the choroid, in moderate abundance in the sclera, and substantially reduced in the retina and RPE. RALDH3 was undetectable in human ocular fundal tissues. In the choroid, RALDH1 and RALDH2 localized to slender cells in the stroma, some of which were closely associated with blood vessels.

Conclusions/Significance

Results of this study demonstrated that: 1) Catalytically active RALDH is present in postnatal human retina, RPE, and choroid, 2) RALDH1 and RALDH2 isoforms are present in these ocular tissues, and 3) RALDH1 and RALDH2 are relatively abundant in the choroid and/or RPE. Taken together, these results suggest that RALDH1 and 2 may play a role in the regulation of postnatal ocular growth in humans through the synthesis of atRA.     

Matrix Metalloproteinases of Normal Human Tissues

This review considers biochemical properties of the family of matrix metalloproteinases (MMPs) of normal human tissues and the involvement of these enzymes in morphogenesis. Four main MMP subfamilies are characterized, and a group of other MMPs is described. Data on mechanisms of activation and inhibition of MMPs in certain tissues during various physiological processes (embryogenesis, angiogenesis, tissue growth and involution) are considered. Information about tissue inhibitors of MMP is presented, and the ability of these inhibitors to regulate the activity of MMPs is analyzed.     

Opposing Growth Regulatory Roles of Protein Kinase D Isoforms in Human Keratinocytes

PKD is a family of three serine/threonine kinases (PKD-1, -2, and -3) involved in the regulation of diverse biological processes including proliferation, migration, secretion, and cell survival. We have previously shown that despite expression of all three isoforms in mouse epidermis, PKD1 plays a unique and critical role in wound healing, phorbol ester-induced hyperplasia, and tumor development. In translating our findings to the human, we discovered that PKD1 is not expressed in human keratinocytes (KCs) and there is a divergence in the expression and function of other PKD isoforms. Contrary to mouse KCs, treatment of cultured human KCs with pharmacological inhibitors of PKDs resulted in growth arrest. We found that PKD2 and PKD3 are expressed differentially in proliferating and differentiating human KCs, with the former uniformly present in both compartments whereas the latter is predominantly expressed in the proliferating compartment. Knockdown of individual PKD isoforms in human KCs revealed contrasting growth regulatory roles for PKD2 and PKD3. Loss of PKD2 enhanced KC proliferative potential while loss of PKD3 resulted in a progressive proliferation defect, loss of clonogenicity and diminished tissue regenerative ability. This proliferation defect was correlated with up-regulation of CDK4/6 inhibitor p15^INK4B and induction of a p53-independent G1 cell cycle arrest. Simultaneous silencing of PKD isoforms resulted in a more pronounced proliferation defect consistent with a predominant role for PKD3 in proliferating KCs. These data underline the importance and complexity of PKD signaling in human epidermis and suggest a central role for PKD3 signaling in maintaining human epidermal homeostasis.