introduction (1).doc

AbstractCarcinoma of the renal parenchyma, also called renal cell carcinoma (RCC) composed of tubular cells in varying arrangements, and is a form of kidney cancer that originates from the proximal renal tubule epithelium. The occurrence of RCC is increasing at a worrying rate and despite the fact that its cure rate is better with the increasingly developed medical technology.It has been reported that agents - Fluorouracil and paclitaxel, would be viable drugs in treating the disease. Attention should be focused on the understanding of the response of RCC to these chemicals. Since the focal obstruction of chemotherapy is multidrug resistance.Considering all these advantages of FTIR, in the past decades, the increasing interest in application of FTIR has been studied. The aim of the present study was to monitor the biochemical changes in renal cell carcinoma (RCC) by Fourier transform infrared spectroscopy (FTIR). The two different kidney cancer cell lines (2245R and 2245R-PAC) were treated by the various concentrations of drugs to determine the optimum way for curing the RCC.The method adopted cultured (RCC) in vitro, the cultured cells applied to CaF2 window film. Then, cell lines were detected by FTIR to obtain a cultured cell line spectrum on the time axis, and the spectral characteristic of these samples were compared. As the corresponding spectral features exist similarities and differences between these cell lines, therefore, this can lay the foundation of laboratory diagnosis of renal cancer.The results of this report suggests that FTIR may be a potential diagnostic tool for cancer chemotherapy monitoring.1. Introduction1.1 RCC1.1.1 Status of RCCMalignant tumor is a serious harm to the physical and mental health of humanbeings and consumes large amounts of medical and health resources. Currently, due to lack of effective treatments for advanced cancer, early diagnosis of cancer has important effects on the prolonging survival time and improving quality of life. In recent years, the incidence of kidney cancer showed a rising trend in the world，increased by about 2.5% per year，worse still, death given rise by RCC is more than 10 million cases annually 1. Kidney cancer is one kind of cancer that begins with the cells in the kidney. RCC is the most common type of kidney cancer, whose occurrence is increasing at a worrying rate and despite the fact that its cure rate is better with the increasingly developed medical technology 2.1.1.2 TreatmentRadical nephrectomy is a greatly effective method for the treating a great majority of RCC patients. Although this is a major surgery, it is a usually a very safe way. For the patients with non-metastatic RCC, the dead rate is mainly less than 1%, whereas this rate is approximately reached to 1% for patients suffering from RCC metastasis. Nephrectomy is an indispensable part during the therapeutic process for people with metasis RCC. Nearly 20% to 40% of those patients, however, merely have survival rate from 35% to 40% and 17% to 30% after 5 and 10 years of surgery, respectively. 3 The situation could be highly bettered through using chemotherapy after the surgery treatment.The uniqueness of chemotherapy is that its effects on the whole human body instead of one area specifically.Thus it also can be called as the systemic treatment, which means drugs impact the region wherever they achieved. Currently, cytotoxic chemotherapy, the primary way for advanced stages of most other solid tumours, however, has virtually no effects on RCC. According to recent studies, there was no single agent for RCC could generate a positive response rate of more than 6% among 50 trials of chemotherapeutic agents 3. Chemotherapy with 5-fluorouracil (5FU) only seems to be effective if combined with immunotherapeutic agents such as Interferon-alpha (IFN-alpha), which provides a promising possibility for curing RCC 2, 4, 5.Studies have shown that, in the treatment of malignant tumors, the role of immune factors become increasingly evident. Since use of immunotherapy help restore immune function and enhance the anti-tumor, reduce relapse of patients, thereby improving survival. Therefore, biological therapy has been combined with chemotherapy by which uses 5-FU plus IFN 10 or IL-2 in combination, a new biological form of kidney cancer chemotherapy treatment, and achieved good results.120 patients out of 215 patients with advanced kidney cancer received biological chemotherapy, and the overall remission rate was 39%, including complete remission (11%) and partial remission (28%) 6.1.1.3 Obstacles of treatmentRCC is not sensitive to chemotherapy and radiotherapy, as biological characteristics of multiple drug-resistant (MDR) is related. MDR refers to cancer cells has drug resisitance to one particular drug, meanwhile, it also works for the other drugs with different structure and targets. MDR was the result of the activity of the drug molecule cells mutate to lift or overexpression. MDR can be divided into two types: (1) natural resistance, namely it reacts in the first chemotherapy ;( 2) the acquired drug resistance, the resistance shows during the process of chemotherapy. 7, 8 MDR is one of the important obstacles affect prognosis kidney cancer ,yet it can be highly bettered along with the development of gene technology and deep study of its a variety of mechanisms ,then selecting high efficiency, low toxicity of chemotherapy sensitive agent.8Antimetabolite is a chemical molecule inhibiting the function of the endogenous metabolite and, generally, these two types of molecules are structurally similar 9, 10. For instance, 5-fluorouracil selectively inhibits the synthesis of DNA or RNA through thymidylate synthase and thymidine synthesis inhibition.The growth of tumor can be stopped as their DNA productino and cell division are interupted by these drugs. The use of 5FU cans interrupt the DNA replication of the cancer cells, which stop the tumor from getting worse. However, the function of antimetabolites would be limited after the MDR has developed to those drugs. In this project, attention is mainly drawing to figure out the MDR to paclitaxel, which is another valuable drug for treating RCC 11.1.2 FTIR1.2.1 Infrared (IR) spectrum The infrared spectrum directly demonstrates that samples absorb red wavelengths of light. It happened when one certain chemical group of a molecule has the same vibrational frequencey as the infrared light, and afterwards the molecule would absorb the energy, changing its groud state to excited state which requring more energy. Different materials have their own uique IR spectrum; therefore, it is quite resonable by interpretation of IR to understand the molecule in detail. 12Normally, the IR is divided into three sections: near-IR area (780-2500nm), intermediate-IR (2500-4000nm) area and far IR areas. For different molecules, the absorbtion frequencey of the same type of groups shows in a relatively small range, and is defined as group frequency. It can be used for the identification of chemical groups, as it shows insignificant alternation despite of the change of molecular configuration. Most of group frequencies locate on the range of 1300-4000 cm-1. And the area from 400 cm-1 to 1330 cm -1 is defined as fingerprint interval, including the finger and group frequency.Regarding to the intensity of IR, regularly, it is denpent on two factors:(1) during the vibration, the more tremendous changes of the dipole moment, the higher the absorbance (2) the possibility of direct energy level transition has positive correlation with absorbance. It should be point out that although every vibration responds to an energy level change, not all these changes would corespondingly generate IR absorbtion spectrum. It is produced only when the instant change of dipole moment occurs 12, 13.1.2.2 The principles of FTIR FTIR is an approach of which is mainly used for gaining an infrared specturm of absortion. A wide range of spectrum can be simultaneously collected by a FTIR spectrometer, containing abundant information of samples. A light source, an interferometer and a detector are the three main parts consists of an FTIR spectrometer, which is showing the following picture.Figure 1: The struture chart of FTIR SpectrometerThe interfometer is the hardcore of the FTIR system. Firstly, the light source is divided into two parts by beam splitter, one reaches to index mirror through transmission, in the meantime, the other one gets to fixed mirror by reflection. Secondly, two beams of light is reflected by index and fixed mirror and come back to beam splitter. The index mirror makes uniform linear motion; as a result, the formation of optical path difference between these two beams of light is created. Finally, light source passes through a sample, the optical information finally shows on ordinary spectra by Fourier transform 14.1.2.3 FTIR application on single cell studyAfter years of deep research on various cancers FTIR sepectrums and getting their characteristic spectral figures, FTIR cancer diagonosis moves from reaserch to clinical stage .The complexity of the tumor tissue causes complex infrared spectrum, indeed, the nature of the difference between benign and malignant tissue is actually from the difference between the cells. Tissue is composed of cells, while the basic polymer of cells is a nucleic acid (DNA, RNA), protein, and phospholipid bilayer membrane. IR spectra of the cells are consisits of the vibration of these polymer molecules. At present, during cancer clinical routine diagnostic procedures, the results are mainly depending on the pathological anatomy and histological diagnosis done by experienced doctors. However, this often causes highly subjective, negative or false positive results 15.FTIR can provide information about changes in the molecular structure and the variety of information on the molecular level and can make changes to reflect the tissue, which is an effective means for early cancer detection. Comparing with traditional means in terms of tumor detection is more accurate, objective and faster. Moreover, the attachment with fiber optic technologies can even sucessuflly achieved the real-time detection and diagonosi in situ tumors in vivo 15, 16.Infrared spectroscopy is mainly rotational spectrum of molecular vibrational spectra and molecular studies to join the bond between atoms. Since 1980s, Fourier transform technique has been applied in infrared spectroscopy, which is an overwholming accheivement. As FTIR has been determined that accquires numerous advantages,such as simple operation porecess, low-noise, high luminous flux,fat meaurement speed, high wavenumber accuracy, wide frequency range, low operating cost and non-destructive testing.Owing to that , it has been widely used in structure, conformational studies of proteins, lipids, carbohydrates and nucleic acids and other biological molecules. On this basis, the comparison of various types of malignant tumors and their corresponding normal tissue or cells of the IR spectral characteristics can help malignant tumors in early diagnosis and open up a new way for the future research development. In this report, attention mainly focusing on Fourier Transform infrared spectral studies of the vitro culture of RCC to lay the cell-based foundation for cancer study 15, 16, 17.1.3 Imaging Conventionally, most microscopes use single point detectors, where the radiation from a small area of sample (defined by the aperture) is focussed onto a small area MCT element resulting in a spectrum. And chemical maps can be produced by moving the samples sequentially in steps in a raster fashion 18. While this mapping is a very slow method of obtaining an image.To solve the problem, an array of detectors is utilised instead of one detector to get the image of the sample so that spectra from each point on the sample can be observed simultaneously,which is called focal plane array.The term “focal plane array” (FPA) refers to an assemblage of individual detector picture elements (“pixels”) located at the focal plane of an imaging system. With light sensors lining up on the focal plane, infrared light from infinity would pass through the optical system and then form images on those sensors. Light signals would be coverted to electrical signals, and then undergoes the integral amplification and sampling hold. These procedures are done by detectors. After these signals pass through the output buffer and multiplex system, eventually, images show on the monitor.The advantages of this new techniques are as follows : (1)a sample to use microscope but not stains or dyes required;(2)contrast in images can be directly collected from the chemical composition of the tissue;(3)each pixel contains information from a frequency bandwidth up to 40 times larger than a visible image.19 ,20 The following picture is a simple demonstration of how do doctors work in the cells (Figure 2). Figure 2: Spectra image gained from applying array of MCT detectors in cells Due to the contrast between the background and detecting temperature would decide the ideal resolution of the detectors, in order to better the precision of the detector the background temperature should be greatly lower. In the project, liquid nitrogen is used for the reducing the temperature.1.4 RMieS-EMSC correctionFTIR is a cheap,simple technique that can be used to evaluate differences in cellular samples.Therefore, after applying the FTIR in the experiment,ideally,the datasets expected from the spectrum are (1)clearly defined band on a flat base line; (2)differences in spectra to represent characteristics of each dataset in biochemistry ; (3)reproducibility at the single cell level.The ideal results, however, are challenging to obtain due to scattering which is a common physical process where some forms of radiation passes through the samples. As a result, the spectrum recorded is very often distorted compared with a pure absorption spectrum of an optimal sample.During the process of data analysis, if scattering not taken into account, any biochemical interpretation of the data should be questionable.Standard ruber band baseline correction is still widely uesed ,yet it is unreliable since it may change the chemical band shape of the specturm.As Mie scattering is one of the highest types of scattering, attention is mainly focused on understanding its origin. Romeo and Diem were the first to fit a Mie scattering curve to an infrared spectrum of a single biological cell and find that it is predominantly from the nucleus of the cell. A modified version, of (Exterded Multiplicative Signal Correction) EMSC (developed by Achim Kohle) will remove the Mie scattering curves, although the broad oscillation resulting from Mie scattering is largely removed, the problem of anomalous dispersion still remains in the spectrum.Presently, there is a developed technology called resonant Mie scattering(RMieS) which can contribute to get the excellent result 15,21 . The following figure 2 can be utilized to fully demonstrate that this technique makes the distastes highly reliable, as it showed that after RMieS corrected distracting dispersions are mostly removed from raw data.Figure 3: Spectra of three renal cancer cell lines In conclusion, the explanation of anomalous dispersion and based distortions in the spectra can be given by RMieS, as it is a correction programme developed to recover the pure absorption spectrum.This enables reliable classification of spectra based on differences in biochemistry.1.5. Data analysisThe necessity of compressing and extracting information in FTIR spectrum is extremely required in qulitative analysis. Firstly, the amount of information containing in spectrums is very large with small absorbance differences among them.Secondly, due to the characterisitic of complexity and variability , the percentage of useful information in FTIR spectrums is relatively small.There are two main ways to solve the problem : principal component analysis and linear discriminant analysis.1.5.1 Principal component analysis (PCA)When making use of statistical method to solve multivarate problems , the complexity of analysis and the amount of caculation would be highly increased as the number of variables is raised.During the process of quantitative analysis, therefore, it would be better that involving less variables and obtaining more useful information. PCA is one functional multivariate statistical analysis method which converts serveral measured variables to less number of uncorrelated index, yet comprehensive. By applying these techniques on this experiment, redundant data can be ignored while the vital data can be left. In addition, it does not require any artificial setting parameters or models which bases on the experience of intervention on the calculation, instead, the final results only related to the data 22.The mathematic principle of principal component analysis method (PCA) lies in coordinate conversion. During the conversion procedure, the given group of related variables are changed into another set of unrelated variables by the means of linear transformation, and these new variables then are arranged according to order of descending variance.The total variance of the variables shoud be keep constant in the mathematical transformation, making the first variable with the biggest variance, which is defined as first principal component. Next, the definition of the second principal components is that its variance is the second large and irrelevent to the first variable. Thus, it can deduce the rest from this, the number of pricipal component actullay denpends on the qualities of variables 22, 23.In the mathematical experssion, the first stag