Vitreous microparticles contain apoptotic signals suggesting a diabetic vitreopathy

INTRODUCTION

Markers used in this study include Annexin-V, CD144, and 4’,6-diamidino-2-phenylindole (DAPI). Annexin-V binds phosphatidyl-serine which is typically found on the inner leaflet of a lipid bilayer. When cells undergo apoptosis,there is a reversal to the polarity of the lipid bilayer and the phosphatidyl serine residues are externalized. Once externalized, Annexin-V can bind and identify a microparticle as Annexin-V-positive. CD144 (V-cadherin) is an endothelial cell marker. DAPI is a membrane impermeable molecule and can pass through a lipid bilayer if it has been compromised.DAPI positive MPs identified by flow cytometry indicate that the MP membrane has been compromised and DAPI has entered the MP. Once inside the MP, DAPI binds nucleic acids whether DNA or RNA.

Microparticles (MPs) are small vesicles bound by a lipid bilayer that pinch off the surface of a cell membrane. The size of MPs range from 0.03-1.0 μm. Exosomes, or membranebound vesicles released by a cell, are formed in the Golgi apparatus or lysosomes, and range in size from 0.03-0.1 μm in diameter; while apoptotic bodies tend to be larger, measuring at the 0.05‐2.0 μm range. MPs consist of fluid and carry a variety of lipid, protein, and nucleic acid cargo including mRNA and micro‐RNAs (miRNA) which can have downstream effects at remote target locations. Baruttadescribed differences in serum miRNAs within extracellular vesicles between patients with diabetes, and found that patients with significantly lower levels of miR-126 within serum extracellular vesicles (EVs)were found to have microvascular complications including diabetic nephropathy. The function of MPs both systemically and within the eye remains an area of intense investigation.There is clear evidence that the pathogenesis of diabetic macular edema and diabetic retinopathy stem from pericyte dysfunction, vascular leakage, and a violation of the bloodretinal-barrier. Extravasation of serum contents into the retina and vitreous may alter the microenvironment of the posterior segment which may lead to exacerbation of retinopathy. Other measures of systemic hyperpermeability such as urinary albumin excretion rate was found to be directly correlated to optical coherence tomography (OCT) macular thickness in patients with diabetes. As such, the presence of serum contents in the posterior segment as a result of retinal vascular hyperpermeability may play a role in disease pathogenesis.

企業(yè)規(guī)范管理的需要。企業(yè)管理不是一個孤立的行為，而是多方面管理措施有機結合最終呈現出來的綜合效果。企業(yè)對各類投入管理的精細化程度反映了該企業(yè)管理水平的高低。企業(yè)管理水平的提高需要各個領域與時俱進，不斷提升?，F代企業(yè)管理越來越規(guī)范化、精細化，越來越重視各類成本的控制。人工成本是企業(yè)總成本的一部分，也需要進行合理控制、精細管理。

Further clinical and basic-science investigations are evaluating the role of the vitreous in various diseases. The Diabetic Retinopathy Clinical Research (DRCR) Network Protocol D evaluated the role of primary vitrectomy in the treatment of diabetic macular edema. In an era when 20-gauge vitrectomy was commonplace and without a consensus regarding the peeling of macular membranes, the outcomes for Protocol D demonstrated that even in patients that had failed conventional therapy for macular edema, up to half of study participants had some short-term improvement in visual acuity and in OCT thickness. This study, however, was a small cohort study that was not designed to address the role of the vitreous in diabetic disease. Protocol AB, which compares prompt vitrectomy to anti-VEGF treatment, will evaluate the role of small-gauge vitrectomy as a primary treatment modality for proliferative diabetic retinopathy. Follow-up for the study is currently underway. The DRIVE-UK study evaluated outcomes following vitrectomy in patients with diabetic eye disease. Patients with mild vitreous hemorrhage treated with vitrectomy had better outcomes than those treated for tractional retinal detachments supporting the concept that the removal of a diseased vitreous may improve future visual outcomes.Vitreous MPs have been evaluated in a few case series.Guptahave evaluated extracellular vesicles using a non-formalin fixative in combination with various imaging techniques including nanoparticle tracking analysis and confocal microscopy. Another group has shown greater concentrations of vitreous MPs and proinflammatory cytokines in patients with rhegmatogenous retinal detachments compared to controls. Exosomes are abundantly expressed in the vitreous. The presence of sub-cellular membrane-derived vesicles in the vitreous with different markers suggests that these vesicles play a role in vitreoretinal pathophysiology. The aim of the current study was two-fold: to optimize the protocol for isolating and identifying human vitreous MPs by flow cytometric analysis, and to identify differences in these MPs between patients with and without diabetes.

The vitreous body fills the posterior segment of the eye and is composed primarily of collagen and hyaluronic acid. These extracellular matrix-molecules are synthesized by hyalocytes, the resident cells of the vitreous. Many disease processes will manifest in the vitreous; for example,uveitis may present secondarily in the form of vitreous cells,or proliferative diabetic retinopathy may present in the form of vitreous hemorrhage or tractional retinal detachment.The vitreous itself, however, has not been evaluated as a primary source for disease pathogenesis. To begin elucidating the role of the vitreous in various diseases, membranebound extracellular vesicles released by hyalocytes have been examined to explore their potential role in disease pathogenesis.

SUBJECTS AND METHODS

This study was approved by the Human Research Protections Office (HRPO) at the Washington University Institutional Review Board (IRB) and adhered to the tenets of the Declaration of Helsinki. Informed consent was obtained from all subjects.

There is convincing evidence that vitreous components play a role in disease pathogenesis relevant to therapy. Physicians use anti-vascular endothelial growth factor (VEGF) agents to treat diverse retinal vascular diseases including diabetic retinopathy and age-related macular degenerationto target elevated levels of VEGF in the posterior segment of the eye. The ability of an intravitreal injection to improve visual outcomes in these patients has revolutionized the way we treat these diseases.As such, the vitreous can serve as a reservoir for diseaseassociated or causal molecules that could potentially be future therapeutic targets or biomarkers.

總之，微小說創(chuàng)作者用兒子“哈哈”大笑的快樂之甚，襯托出了單親媽媽“嚎啕大哭”的哀傷之深。這種以“樂景寫哀情”的表達技巧，被創(chuàng)作者貼切地運用到微小說《等》中，可謂獨具匠心！它對于深化作品主題——在歌頌母愛和贊揚母親的偉大的同時，對現實生活中的那些不理解父母一生艱辛、成家立業(yè)后仍在“傍老”“啃老”的子女，給予行為上的批判和道德上的譴責，無疑起了非常重要的作用。

Inclusion criteria included eyes undergoing primary vitrectomy for any disease process except uveitis. A chart review was performed to acquire historical data such as presence and absence of diabetes mellitus and the reason for surgery. The patients underwent standard 3-port trans-conjunctival vitrectomy with sterile precautions. Three 25-gauge trocars were inserted into the eye and the vitrector was inserted into the mid-vitreous cavity. An assistant would aspirate the vitreous into a 3 mL syringe to an appropriate volume prior to the visualization of choroidals or ocular collapse. For the initial 9 patients, cut rates varied between 1000 cuts per minute (cpm) to 7500 cpm. Subsequent to this,future specimens were cut at 1000 cpm. Specimens were transferred into freezer-appropriate 3 mL vials and immediately placed on dry ice and later placed into a -80°C freezer.

In order to optimize MP signal for flow cytometry, it was important to titrate parameters to attain the best possible signal. This signal is the ability for the cytometer to identify a single MP, as opposed to two MPs(doublets) that may be stuck together. When a cytometer reads a doublet, the amount of fluorescence emitted by that particle is therefore an aberrant reading since two MPs would have more fluorescence together than a single MP alone and can alter specimen interpretation. Singlets were defined as a population of particles within a 20K-unit difference in sidescatter-width that may span a range of side-scatter-area-levels,while doublets and other multiplets were particles with greater side-scatter-width values. Vitrectomy cut rates were titrated between 1000 and 7500 cpm to identify cut rates with optimal singlet yield.

由于各高校實際情況的不同，實驗班體制的設定也有很大差異。根據具體高校的教育資源和師資力量的不同，實驗班的開展主要采取具體的實體班級制度或虛擬班級制度。以湖南大學為例，實體班級制度與前文的實驗班的開展略有差別，由學生所在院系和合作院系共同管理，與前文實驗班的管理模式差別不大不作贅述；其中虛擬班級制度則是在學生入校之初便按照所報專業(yè)作為主專業(yè)進入院系學習，由主專業(yè)所在院系教學。學生的課程學習則是按照個人傾向的第二專業(yè)，由第二專業(yè)所在的院系負責課程方案的設定。

Annexin‐V binding media and resuspended in a final 200 μL of Annexin‐V binding media with DAPⅠ prior to flow cytometry.DAPI-control-specimens were permeabilized with 100% icecold methanol at 4°C for 20min.

正確的做法，應該是“丑話說在前面”。賽季前，理當公開向媒體并通過媒體向廣大球迷講明白俱樂部遇到的難處，著重強調球隊在人員不整、備戰(zhàn)倉促情況下面對的嚴峻形勢，給輿論降溫。賽季目標定為“力爭季后賽”，同時說清楚，球隊本賽季的工作重心是打基礎，重用新人，重塑戰(zhàn)術體系，成績是第二位的。拉低社會期望值，就給俱樂部和球隊的東山再起爭取到了最寶貴的時間和空間。

To evaluate MP membrane integrity, non-frozen specimens were evaluated per protocol above with incubation only with DAPI. Specimens were divided into two aliquots and the second aliquot was evaluated the subsequent day. Specimens were kept at 4°C overnight and evaluated the subsequent day for DAPI fluorescence.

Samples were thawed at 37°C for 5min and transferred to tissue culture plates with Dulbecco’s modified Eagles medium (DMEM) with 10% fetal bovine serum, 1% penicillin, 1% streptomycin and 2 mg/mL type II collagenase. The tissue culture plate was placed in a 37°C shaker for 1h rotating at 100 rotations per minute. MPs were then collected per recommendations by the International Society of Thrombosis and Hemostasis. In detail, specimens were centrifuged at 2500 g for 15min. The supernatant was aspirated and resuspended in 1 mL of phosphate buffered saline (PBS) and centrifuged again at 2500 g. The supernatant was aspirated and specimens were resuspended in 100 μL of PBS with mouse monoclonal anti-human CD45 antibody conjugated to fluorescein isothiocyanate(FITC, Thermo Fisher) and mouse monoclonal anti-human CD144 (V-cadherin) antibody conjugated to allophycocyanin(APC, Thermo Fisher) for 30min on ice. Specimens were washed with PBS and centrifuged at 2500 g for 15min. The supernatant was aspirated and the pellet resuspended in Annexin-V-binding-media. The specimen was again spun down at 2500 g for 15min. The supernatant was aspirated and the pellet resuspended in 100 μL of Annexin-V-binding media with Annexin-V bound to phycoerythrin (PE) and incubated for 15min at room temperature. Specimens were washed with

A BD LSR II flow cytometer was used for analysis. FITC, PE, and APC compensation beads were used as positive controls for each fluorophore.Prior methanol permeabilized specimens served as positive DAPI controls. The forward scatter laser is a 488 nm laser establishing the lower threshold for particle detection just under 0.5 μm. Sub-micron beads ranging from 0.5-2.0 μm were used for size-comparison. Gating was initially focused on obtaining singlet microparticle populations. Subsequent gating was performed on DAPI negative specimens to evaluate specimens with intact lipid bilayers. Further analysis for percentages of CD45, CD144, and Annexin-V positivity were evaluated with histogram or graphical functions. FlowJo 10(FlowJo for Windows, Version 10, FlowJo LLC) was used for analysis.

Due to their small and variable sizes, many methods have been devised to best extract and isolate exosomes and EVs from various tissues.The centrifugation method used in this study was that recommended by the International Society of Thrombosis and Hemostasis. MP-isolation from vitreous presented an additional challenge compared to serum protocols due to the presence, in vitreous, of overlying extracellular matrix-tissue.To address this, vitreous specimens were thereby digested with type II collagenase in tissue culture media for 1h prior to centrifugation.

RESULTS

In order to obtain the highest yield of MP singlets, vitrectomy cut rates were altered in order to determine the effect on yield. A low cut rate was established at 1000 cpm and higher cut rates at 4000 and 7500 cpm. Due to differences in vitrector manufacturing,cutting mechanisms vary. The Alcon Constellation25-gauge vitrector cuts once per cut: it cuts as the cutting-apparatus moves towards the tip of the vitrector, but not when it moves back towards the handpiece. The DORC Evasystem,however, cuts twice per cut, once in each direction as the cutter passes away, towards the tip of the vitrector, and again when the cutting-apparatus returns towards the handpiece. Specimens collected by the Eva-vitrectomy system were collected to approximate the effective cut rate of the Constellation‐system(therefore a 500 cpm Eva-collected specimen was equivalent to a 1000 cpm Constellation-collected specimen). Specimens obtained at a lower cut rate yielded 92%±5.5% (mean±SD,=4) singlets per specimen whereas those obtained at higher cut rates yielded 66%±12.8% (=5,=0.016; Figure 1). After the analysis of these 9 specimens, subsequent specimens were collected at the 1000 cpm cut-rate and these former specimens were not included in subsequent analysis.

This study aimed to establish methods to improve signal quality for performing flow cytometry on human vitreous MP populations. In addition, differences in MP profiles between patients with and without diabetes who have undergone vitrectomy were also established during this study.

在運用該系統(tǒng)的過程最后，車輛進場前，需在項目部安全環(huán)保部門辦理臺賬登記手續(xù)（三證齊全），手續(xù)齊全后發(fā)項目部自編號，由設備物資科進行電子標簽的錄入信息工作，錄入完成后裝料，裝料到達自動稱重系統(tǒng)后看指示燈（紅綠燈），當綠燈亮時，車輛行駛到電子標簽掃描區(qū)域，掃描成功后道閘打開，車輛進入稱臺中間（此時紅外線已掃描）不用停車方可稱重記錄，記錄成功后語音提示稱重保存成功，顯示屏顯示稱重數據，道閘打開車輛通過稱重完畢。

Certain disease processes were found to inherently have greater percentages of MPs than others among fresh specimens.Patients with macular holes or epiretinal membranes were found to have 56%±7.1% (=3) singlets per fresh specimen compared to 88%±8.6% (=6) in patients with diabetic eye disease such as non-clearing vitreous hemorrhage due to proliferative retinopathy or tractional retinal detachment(=0.024; Figure 3).

A representative flow plot is shown in Figure 4A demonstrating a specimen from a macular hole surgery while Figure 4B demonstrates a specimen from a tractional retinal detachment surgery with differences in singlet yields. Once singlets were identified, attention was drawn to DAPⅠ negative populations(MPs with intact membranes), and Annexin-V and CD144 positivity (APC and PE‐fluorophores respectively), as shown in Figure 5.

Patient baseline characteristics are shown in Table 1. Patients undergoing vitrectomy for diabetic eye disease such as nonclearing vitreous hemorrhage or tractional retinal detachment have infiltration of whole blood into the vitreous. This alters the MPs in the vitreous in that there are blood-derived MPs in the vitreous in addition to hyalocyte-derived MPs. To explore this, we collected specimens from patients with diabetes who underwent vitrectomies unrelated to their diabetes for conditions such as macular hole repair or rhegmatogenous retinal detachment (=8). We also collected specimens from patients without diabetes who developed non-clearing vitreous hemorrhage from another cause, such as a retinal tear with a torn bridging retinal vessel (=5). A representative flow plot demonstrating Annexin-V positivity between MPs from a macular hole surgery compared to a diabetic non-clearing vitreous hemorrhage is shown in Figure 6.

The presence of Annexin-V on MPs from patients with and without diabetes was not significantly different overall(39.0%±33.8%,=1524.9%±30.1%,=19, respectively;=0.228). However, MPs from samples with vitreous hemorrhage had greater Annexin-V positivity than MPs from samples without hemorrhage (52.3%±30.7%19.6%±27.2%,respectively,=0.002; Figure 7). Amongst patients with diabetes, MPs from hemorrhagic samples had greater Annexin-V positivity compared to those without hemorrhage(62.1%±31.7%,=718.9%±20.9%,=7,=0.009; Figure 8).In contrast, in samples obtained from patients without diabetes,Annexin-V positivity on MPs in vitreous hemorrhage was not different from other disease processes (38.6%±25.7%,=520.0%±30.9%,=14,=0.087; Figure 8). Taken together,this data suggests that the presence of Annexin-V positivity, a marker of apoptosis, is a feature of diabetic hemorrhage.

第四，形成簡單、自然的分工協作。在閩商的行銷網絡里，在團隊協作中，每個人都能自然、自覺地找到自己的位置，發(fā)揮自己的作用，得到自己付出的酬勞。有錢的當老板、沒錢的做伙計；有的批發(fā)、有的零售；有的行商推銷，有的組織生產供貨、有的提供金融服務；有的搞運輸、有的跑業(yè)務…雖然沒有人指揮，卻能做到既分工明確，又內外協調，井然有序。

The endothelial marker CD144 or V-cadherin was analyzed and shown to have greater positivity in vitreous with hemorrhage than without hemorrhage (18.2%±22.4%,=70.8%±1.7%,=12, respectively,=0.007). In the subset of patients with diabetes, this difference was not statistically significant (10.8%±17.5%,=31.8%±2.5%,=5,=0.786).When evaluating patients without diabetes, those with hemorrhage maintained elevated levels of CD144 compared to other disease processes (23.8%±26.5%,=4,0.02%±0.03%,=7, respectively,=0.006; Figure 9). This finding suggests a baseline vascular leakage in non-hemorrhagic diabetic specimens and confirming greater levels of endothelial‐derived MPs in specimens with hemorrhage.

只有老太醫(yī)和喬十二郎兩人內心清楚：那天李武崗喝下的海狗油，有股特別的香氣，那不是海狗油，是效力強大的燃油和火藥；那天吸附在李武崗后背上的十二只火罐，每只罐底外部都額外加了點燃的艾絨，名曰增強藥力，其實那是構造奇特的引信。

DISCUSSION

To evaluate sample stability, membrane integrity was assessed by DAPI exclusion. Fresh MP-specimens were evaluated the day of collection without a freeze-thaw cycle and divided into two aliquots. The first aliquot was examined the same day of collection after incubation with DAPI. The second aliquot was kept at 4°C overnight and evaluated the next day. Specimens evaluated the day of collection demonstrated a lower DAPI fluorescence than the subsequent day, suggesting a decrease in membrane integrity with time. However, difference was not found to be statistically significant in the 3 specimens in which this was performed (=0.106; Figure 2).

Statistical analysis was performed using SPSS statistical software (SPSS for Windows, Version 23.0;ⅠBM‐SPSS). Significance was defined with<0.05. As this was an exploratory study, sample size calculations were not performed.Continuous data sets between two groups were compared using the Mann-Whitneytest and paired samples at two time points were analyzed using the Wilcoxon-Rank-Sum test.

Methods for optimizing MP signal for flow cytometric analysis are also described. Reducing cut rate to 1000 cpm compared to the general maximum cut rates of 7500-8000 cpm may preserve MPs integrity and reduce debris formation which may occur with faster cut rates. We noted only minimal loss of MP membrane integrity during storage at 4°C overnight as evidenced by an increase in DAPI fluorescence on the subsequent day. While the change in membrane integrity was not significantly different in 3 samples, this finding indicates that evaluating fresh specimens is preferable, as overnight storage does degrade specimen quality. Subsequent specimens in this study, however, were evaluated after being frozen.The signal strength (percent of MP singlets in a sample) was found to be greater in certain diseases such as diabetic nonclearing vitreous hemorrhage and tractional retinal detachment compared to other surgeries such as rhegmatogenous retinal detachment, macular hole surgery, or epiretinal membrane surgery.This may be partly due to blood having entered the vitreous and contamination of vitreous MPs by whole blood MPs. An increase in total MPs by blood MPs would improve the singlet population of MPs found in the vitreous and thereby signal strength.

EVs and MPs are released by nearly all cell types and play a role in physiologic conditions such as cellular activationand apoptosisto pathologic conditions including malignant transformationand inflammation. The role of vitreous MPs in the pathogenesis of diabetic retinopathy, however, has not been characterized.

Endothelial and pericyte dysfunction play a clear role in the pathogenesis of diabetic retinopathy and the leakage of vascular contents into the retina. Intact tight junctions within retinal endothelium maintain the blood-retina barrier keeping out various antigenic stimuli, macromolecules and serum MPs. Endothelial dysfunction in diabetes, however, can result in the production of pathologic MPs which contribute to the inflammatory and atherogenic responseas well as leakage of serum contents into the retina and vitreous.Circulating serum MPs have been evaluated in patients with varying levels of glucose tolerance demonstrating a gradual increase in Annexin-VMPs in the serum of patients with greater glucose intolerance.

Patients with vitreous hemorrhage were found to have greater levels of Annexin-VMPs than those without vitreous hemorrhage. This difference, however, was specific to diabetic vitreous hemorrhage, and not vitreous hemorrhage explained by other disease processes such as a torn retinal vessel in the setting of a retinal tear or detachment. Increased Annexin-V positivity in serum MPs have been previously described in patients with diabetes by Giannella. Extravasation of serum contents, including pro-apoptotic MPs, into the posterior segment may play a role in the progression of diabetic retinopathy. Methods to dampen the extravasation of serum contents may help improve outcomes. One pathway that treats vascular hyper-permeability, the Tie2/Angiopoietin pathway, has been shown in clinical trials to treat diabetic macular edema and age-related macular degeneration and to help improve outcomes. Inhibition of such pathways may reduce the extravasation of these pro-apoptotic markers into the vitreous, however these functional assays have yet to be performed.

Eyes containing vitreous hemorrhage also had greater levels of CD144MPs than those without hemorrhage. Chaheddescribe increased CD144MPs in patients undergoing vitrectomy for proliferative diabetic retinopathy, however this may be secondary to vascular leakage and introduction of serum MPs into the vitreous rather than being pathology related to diabetes. Evidence is provided in this study showing that vitreous hemorrhage even in patients without diabetes contains elevated levels of CD144MPs. This is not a surprising finding as most MPs contain markers from their cell of origin; and MPs from the vasculature would contain elevated levels of CD144 which is derived from endothelium.The presence of an abnormal MP-environment within the vitreous may play a role in the worsening of diabetic retinopathy. The DRCR.net Protocol AB may show how early vitrectomy may compare to anti-VEGF treatment alone, and to see if removal of a diseased vitreous may improve visual outcomes. Vascular leakage and breakdown of the bloodretina-barrier and introduction of serum MPs may contribute to worsening retinopathy. The removal of these diseased MPs may improve outcomes. Vitreomacular adhesions are associated with worse diabetic retinopathy outcomes, supporting the notion that the vitreous body may promote disease in patients with diabetes. The findings of the present study could represent one mechanism that partially accounts for such an effect.In conclusion, vitreous MPs reflect changes seen in progressive diabetic retinopathy including those seen with vitreous hemorrhage and proliferative diabetic retinopathy.The presence of blood in the vitreous of patients with diabetes may contribute to a diseased vitreous by evidence of increased Annexin-VMPs, a sign of cellular apoptosis. Significant work is needed to further understand the role and function of MPs in the vitreous and how they may contribute to progression of diabetic eye disease.

Limitations to this study include the relatively small sample sizes in group comparisons. This study is also un-masked which may bias the results. Limitation in sample collection includes the variability in aspiration. Standardized aspiration rates have not been established as it is an assistant that collects the specimen in the 3 mL syringe. Despite establishing a vitrectomy cut‐rate, person‐to‐person differences in aspiration rate can also change MP singlet populations which was not controlled in this study.Most of the current analysis was done on vitreous specimens that were frozen. The current microparticle literature demonstrates that freezing MPs may alter MP integrity and artifactually increase the number of MPs seen by flow cytometry known as MP-fracture. Due to the labor intensiveness of processing and evaluating samples the same day as sample collection, the majority of samples were still evaluated with a single freeze-thaw cycle, and thus the results should be interpreted with caution. A significant amount of clinical MP literature, however, supports a protocol where specimens have undergone a single freeze-thaw cycle.

The BD LSR ⅠⅠ flow cytometer has a lower limit of detection at 0.488 μm, the wavelength of its forward scatter laser. This study only evaluates MPs that are between detectable range through 2.0 μm but not those that are smaller, in the 0.03 μm range. This study may bias its results towards MPs that are larger and neglecting smaller MPs that are beyond the lower detection-limit of the cytometer. Sample size calculations were not done to make the conclusions seen in the study and thus must be considered exploratory.

在“116號文”中，建設單位臨時房屋建筑劃歸在第四部分臨時工程中，是和承包人的臨時辦公、生活、文化福利建筑合并在一起計算的。這種規(guī)定不甚恰當，一是建設單位不知道自己在臨時房屋建筑投資上的利益及責任，無以控制其建設規(guī)模、標準和考核投資效果；二是不利于招標投標時各方計價，不利于招標投標價與設計概算的對比分析。

Supported by an unrestricted grant to Washington University by Research to Prevent Blindness Inc., New York, NY.

擬建建筑物基坑開挖2.0～4.8 m，距離建筑物的最小距離約為30 m，西側主要為道路，無重要建(構)筑物及大型市政管線分布，基坑側壁安全等級為三級。

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